Methods for predicting drug responsiveness in cancer patients

ABSTRACT

The present invention features methods, devices, and kits for detecting expression in a patient having cancer or determining responsive of a patient having cancer to a treatment, such as irofulven. The invention further includes methods of treating a patient having cancer by administering, e.g., irofulven.

FIELD OF THE INVENTION

The invention features methods to detect the expression levels of genesencoding biomarkers in cancer patients and to predict the responsivenessof cancer patients to irofulven.

BACKGROUND

DNA microarrays have been used to measure gene expression in samplesfrom patients and to facilitate diagnosis of disease. Gene expressioncan reveal the presence of cancer in a patient in addition to the type,stage, and origin of the cancer. Gene expression may even have a role inpredicting the efficacy of cancer therapies. In recent decades, theNational Cancer Institute (NCI) has tested cancer therapeutics for theireffect in limiting the growth of 60 human cancer cell lines. The NCI hasalso measured gene expression in those 60 cancer cell lines using DNAmicroarrays. Various studies have explored the relationship between geneexpression and therapeutic effect using the NCI datasets.

During cancer treatment, critical time is often lost due to a trial anderror approach to finding an effective therapy. In addition, cancercells often develop resistance to a previously effective therapy. Insuch situations, patient outcome would be greatly improved by earlydetection of such resistance.

Thus, there exists a need in the art for methods and devices that canpredict the responsiveness of cancer patients to a medical treatment.

SUMMARY OF THE INVENTION

The invention features methods for detecting expression of a biomarker(e.g., one, two, three, four, five, ten, twenty, or all of thebiomarkers shown in Tables 1-4, such as ATP1B1 (SEQ ID NO: 201)) in apatient having cancer, such as a patient having cancer (e.g., a human)that is resistant to one or more cancer therapies other than irofulven(e.g., a patient with prostate cancer, ovarian cancer, or hepatocellularcarcinoma (HCC) that is resistant to one or more cancer therapies otherthan irofulven), and for determining responsiveness of a cancer patient(e.g., a patient with prostate cancer, ovarian cancer, or HCC) totreatment with irofulven. The invention also features methods oftreating cancer in a patient in need thereof (e.g., a patient withprostate cancer, ovarian cancer, or HCC or a treatment resistant formthereof) that include administering irofulven to the patient, in whichthe patient is or has been determined to be responsive to irofulvenaccording to the diagnostic methods described herein.

Exemplary types of cancer that can be diagnosed or treated with themethods include, e.g., prostate cancer, ovarian cancer (e.g., ovarianadenocarcinoma or embryonal carcinoma), liver cancer (e.g., HCC orhepatoma), myeloma (e.g., multiple myeloma), colorectal cancer (e.g.,colon cancer and rectal cancer), leukemia (e.g., acute myeloid leukemia,acute lymphoid leukemia, chronic myeloid leukemia, chronic lymphocyticleukemia, acute myeloblastic leukemia, acute promyelocytic leukemia,acute myelomonocytic leukemia, acute monocytic leukemia, acuteerythroleukemia, and chronic leukemia), myelodysplastic syndrome,lymphoma (e.g., diffuse large B-cell lymphoma, cutaneous T-celllymphoma, peripheral T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin'slymphoma, Waldenstrom's macroglobulinemia, and lymphocytic lymphoma),cervical cancer, esophageal cancer, melanoma, glioma (e.g.,oligodendroglioma), pancreatic cancer (e.g., adenosquamous carcinoma,signet ring cell carcinoma, hepatoid carcinoma, colloid carcinoma, isletcell carcinoma, and pancreatic neuroendocrine carcinoma),gastrointestinal stromal tumor, sarcoma (e.g., fibrosarcoma,myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma,angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, leiomyosarcoma, Ewing's sarcoma, andrhabdomyosarcoma), breast cancer (e.g., medullary carcinoma),ER-positive cancer, bladder cancer, head and neck cancer (e.g., squamouscell carcinoma of the head and neck), lung cancer (e.g., non-small celllung carcinoma, large cell carcinoma, bronchogenic carcinoma, andpapillary adenocarcinoma), metastatic cancer, oral cavity cancer,uterine cancer, testicular cancer (e.g., seminoma and embryonalcarcinoma), skin cancer (e.g., squamous cell carcinoma, and basal cellcarcinoma), thyroid cancer (e.g., papillary carcinoma and medullarycarcinoma), brain cancer (e.g., astrocytoma and craniopharyngioma),stomach cancer, intra-epithelial cancer, bone cancer, biliary tractcancer, eye cancer, larynx cancer, kidney cancer (e.g., renal cellcarcinoma and Wilms tumor), gastric cancer, blastoma (e.g.,nephroblastoma, medulloblastoma, hemangioblastoma, neuroblastoma, andretinoblastoma), polycythemia vera, chordoma, synovioma, mesothelioma,adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma,cystadenocarcinoma, bile duct carcinoma, choriocarcinoma, epithelialcarcinoma, ependymoma, pinealoma, acoustic neuroma, schwannoma,meningioma, pituitary adenoma, nerve sheath tumor, cancer of the smallintestine, cancer of the endocrine system, cancer of the penis, cancerof the urethra, cutaneous or intraocular melanoma, a gynecologic tumor,solid tumors of childhood, or neoplasms of the central nervous system.For example, the cancer may be a solid tumor or a hematological cancer.

A first aspect of the invention features a method for detectingexpression of a biomarker (e.g., one, two, three, four, five, ten,twenty, or all of the biomarkers shown in Tables 1 and/or 2, such asATP1B1 (SEQ ID NO: 201)) in a patient having cancer (e.g., prostatecancer, ovarian cancer, or HCC), such as a patient having cancer that isresistant to one or more cancer therapies other than irofulven (e.g., apatient with prostate cancer, ovarian cancer, or HCC that is resistantto one or more cancer therapies other than irofulven). The methodincludes (a) contacting a sample (e.g., a tumor sample) from the patientincluding one or more nucleic acid molecules with a device (e.g., amicroarray) including: i) one or more single-stranded nucleic acidmolecules capable of specifically hybridizing with the nucleotides ofone or more biomarkers of sensitivity selected from the biomarkers ofTables 1 and 3 (e.g., ATP1B1 (SEQ ID NO: 201)); and/or ii) one or moresingle-stranded nucleic acid molecules capable of specificallyhybridizing with the nucleotides of one or more biomarkers of resistanceselected from the biomarkers of Tables 2 and 4 (e.g., IGLC1 (SEQ ID NO:301, 302, 303, or 318)); and (b) detecting a level of expression of oneor more of the biomarker(s) of sensitivity and/or one or more of thebiomarkers of resistance by performing microarray analysis orquantitative reverse transcriptase polymerase chain reaction (qRT-PCR).Expression of the biomarker(s) may be detected by determining the levelof a messenger RNA (mRNA) transcribed from a nucleic acid moleculecorresponding to a gene of the biomarker(s; e.g., a mRNA expressed fromthe ATP1B1 gene (SEQ ID NO: 201)) or a complementary DNA (cDNA) thereof.

A second aspect of the invention features a method of determiningresponsiveness of a patient having cancer (e.g., one of the cancersnoted above, such as prostate cancer, ovarian cancer, or HCC) toirofulven. In particular, the patient may have a cancer that isresistant to one or more cancer therapies other than irofulven, such asprostate cancer, ovarian cancer, or HCC that is resistant to one or morecancer therapies other than irofulven. The method includes a) contactinga sample (e.g., a tumor sample) from the patient including one or morenucleic acid molecules with a device (e.g., a microarray or a device forperforming a qRT-PCR reaction) including: i) one or more single-strandednucleic acid molecules capable of specifically hybridizing with thenucleotides of one or more biomarkers of sensitivity selected from thebiomarkers of Tables 1 and 3 (e.g., ATP1B1 (SEQ ID NO: 201)); and/or ii)one or more single-stranded nucleic acid molecules capable ofspecifically hybridizing with the nucleotides of one or more biomarkersof resistance selected from the biomarkers of Tables 2 and 4 (e.g.,IGLC1 (SEQ ID NO: 301, 302, 303, or 318)); and b) measuringhybridization, or an amplification product resulting from hybridization,between the one or more nucleic acid molecules from the patient and thesingle-stranded nucleic acid molecules of the device to detect a levelof expression of one or more of the biomarkers of sensitivity and/or oneor more of the biomarkers of resistance. The patient is determined to beresponsive to irofulven if: i) the level of expression of the biomarkersof sensitivity is substantially similar to the level of expression ofthe biomarkers of sensitivity (e.g., ATP1B1 (SEQ ID NO: 201)) in a cell(e.g., a cancer cell) or tissue (e.g., a tumor tissue) known to besensitive to irofulven; and/or ii) the level of expression of thebiomarkers of resistance (e.g., IGLC1 (SEQ ID NO: 301, 302, 303, or318)) is substantially dissimilar to the level of expression of thebiomarkers of resistance in a cell (e.g., a cancer cell) or tissue(e.g., a tumor tissue) known to be resistant to irofulven.

The method of the second aspect can further include administeringirofulven to the patient if: i) the level of expression of thebiomarkers of sensitivity (e.g., ATP1B1 (SEQ ID NO: 201)) issubstantially similar to the level of expression of the biomarkers ofsensitivity in a cell (e.g., a cancer cell) or tissue (e.g., a tumortissue) known to be sensitive to irofulven; and/or ii) the level ofexpression of the biomarkers of resistance (e.g., IGLC1 (SEQ ID NO: 301,302, 303, OR 318)) is substantially dissimilar to the level ofexpression of the biomarkers of resistance in a cell (e.g., a cancercell) or tissue (e.g., a tumor tissue) known to be resistant toirofulven. The method can further include administering one or morecancer therapies other than irofulven to the patient if: i) the level ofexpression of the biomarkers of sensitivity (e.g., ATP1B1 (SEQ ID NO:201)) is substantially dissimilar to the level of expression of thebiomarkers of sensitivity in a cell (e.g., a cancer cell) or tissue(e.g., a tumor tissue) known to be sensitive to irofulven; and/or ii)the level of expression of the biomarkers of resistance is substantiallysimilar to the level of expression of the biomarkers of resistance(e.g., IGLC1 (SEQ ID NO: 301, 302, 303, OR 318)) in a cell (e.g., acancer cell) or tissue (e.g., a tumor tissue) known to be resistant toirofulven. In particular, the one or more of the cancer therapiesincludes surgery, radiation, or a therapeutic agent, such as adocetaxel, cabazitaxel, mitoxantrone, estramustine, prednisone,carboplatin, bevacizumab, paclitaxel, gemcitabine, doxorubicin,topotecan, etoposide, tamoxifen, letrozole, sorafenib, fluorouracil,capecitabine, oxaliplatin, interferon-alpha, 5-fluorouracil (5-FU), ahistone deacetylase (HDAC) inhibitor, ipilimumab, bortezomib,carfilzomib, thalidomide, lenalidomide, pomalidomide, dexamethasone,cyclophosphamide, vincristine, melphalan, tegafur, irinotecan,cetuximab, leucovorin, SN-38, everolimus, temsirolimus, bleomycin,lomustine, depsipeptide, erlotinib, cisplatin, busulfan, epirubicin,arsenic trioxide, bendamustine, fulvestrant, teniposide, adriamycin,decitabine, estramustine, azaguanine, aclarubicin, mitomycin,paclitaxel, taxotere, APO010, ara-c, methylprednisolone, methotrexate,methyl-gag, belinostat, idarubicin, IL4-PR38, valproic acid, all-transretinoic acid (ATRA), cytoxan, suberoylanilide hydroxamic acid,leukeran, fludarabine, vinblastine, dacarbazine, hydroxyurea, tegafur,daunorubicin, mechlorethamine, streptozocin, carmustine, mercaptopurine,dactinomycin, tretinoin, ifosfamide, floxuridine, thioguanine, PSC 833,herceptin, celecoxib, iressa, anastrozole, and rituximab.

The invention also features a method of treating cancer in a patient inneed thereof (e.g., one of the cancers noted above, such as prostatecancer, ovarian cancer, or HCC) that includes administering irofulven tothe patient, in which the patient has been determined to be responsiveto irofulven according to the method of the first or second aspect ofthe invention. In particular, the patient may have a cancer that isresistant to one or more cancer therapies other than irofulven (e.g., apatient with prostate cancer, ovarian cancer, or HCC that is resistantto one or more cancer therapies other than irofulven).

A third aspect of the invention features a method of treating a patienthaving cancer (e.g., one of the cancers noted above, such as prostatecancer, ovarian cancer, or HCC). In particular, the patient may have acancer that is resistant to one or more cancer therapies other thanirofulven (e.g., a patient with prostate cancer, ovarian cancer, or HCCthat is resistant to one or more cancer therapies other than irofulven).The method includes a) contacting a sample (e.g., a tumor sample) fromthe patient including one or more nucleic acid molecules with a device(e.g., a microarray or a device for performing a qRT-PCR reaction)including: i) one or more single-stranded nucleic acid molecules capableof specifically hybridizing with the nucleotides of one or morebiomarkers of sensitivity selected from the biomarkers of Tables 1 and 3(e.g., ATP1B1 (SEQ ID NO: 201)); and/or ii) one or more single-strandednucleic acid molecules capable of specifically hybridizing with thenucleotides of one or more biomarkers of resistance selected from thebiomarkers of Tables 2 and 4 (e.g., IGLC1 (SEQ ID NO: 301, 302, 303, OR318)); and b) measuring hybridization, or an amplification productresulting from hybridization, between the one or more nucleic acidmolecules from the patient and the single-stranded nucleic acidmolecules of the device to detect a level of expression of one or moreof the biomarkers of sensitivity and/or one or more of the biomarkers ofresistance; and c) administering irofulven to the patient if: i) thelevel of expression of the biomarkers of sensitivity is substantiallysimilar to the level of expression of the biomarkers of sensitivity in acell (e.g., a cancer cell) or tissue (e.g., a tumor tissue) known to besensitive to irofulven; and/or ii) the level of expression of thebiomarkers of resistance is substantially dissimilar to the level ofexpression of the biomarkers of resistance in a cell (e.g., a cancercell) or tissue (e.g., a tumor tissue) known to be resistant toirofulven.

The method of the third aspect of the invention may further includeadministering one or more additional therapies (e.g., surgery,radiation, or a therapeutic agent) to the patient prior to, concurrentlywith, or after administration of irofulven. In particular, thetherapeutic agent may be selected from the group consisting ofdocetaxel, cabazitaxel, mitoxantrone, estramustine, prednisone,carboplatin, bevacizumab, paclitaxel, gemcitabine, doxorubicin,topotecan, etoposide, tamoxifen, letrozole, sorafenib, fluorouracil,capecitabine, oxaliplatin, interferon-alpha, 5-fluorouracil (5-FU), ahistone deacetylase (HDAC) inhibitor, ipilimumab, bortezomib,carfilzomib, thalidomide, lenalidomide, pomalidomide, dexamethasone,cyclophosphamide, vincristine, melphalan, tegafur, irinotecan,cetuximab, leucovorin, SN-38, everolimus, temsirolimus, bleomycin,lomustine, depsipeptide, erlotinib, cisplatin, busulfan, epirubicin,arsenic trioxide, bendamustine, fulvestrant, teniposide, adriamycin,decitabine, estramustine, azaguanine, aclarubicin, mitomycin,paclitaxel, taxotere, APO010, ara-c, methylprednisolone, methotrexate,methyl-gag, belinostat, idarubicin, IL4-PR38, valproic acid, all-transretinoic acid (ATRA), cytoxan, suberoylanilide hydroxamic acid,leukeran, fludarabine, vinblastine, dacarbazine, hydroxyurea, tegafur,daunorubicin, mechlorethamine, streptozocin, carmustine, mercaptopurine,dactinomycin, tretinoin, ifosfamide, floxuridine, thioguanine, PSC 833,herceptin, celecoxib, iressa, anastrozole, and rituximab. Thetherapeutic agent can be administered parenterally (e.g. intravenously,intramuscularly, transdermally, intradermally, intra-arterially,intracranially, subcutaneously, intraorbitally, intraventricularly,intraspinally, intraperitoneally, or intranasally), enterally, ortopically.

In the second or third aspect of the invention, irofulven may beadministered parenterally (e.g. intravenously, intramuscularly,transdermally, intradermally, intra-arterially, intracranially,subcutaneously, intraorbitally, intraventricularly, intraspinally,intraperitoneally, or intranasally), enterally, or topically.Preferably, irofulven is administered by intravenous injection.Irofulven may be administered to the patient two or more times, such asone or more times daily, weekly, every two weeks, every three weeks, ormonthly (e.g., one or more times per week for two weeks or more).Additionally, a second dose of irofulven may be administered to thepatient two weeks, three weeks, four weeks, or five weeks afteradministration of a prior dose of irofulven. In particular, irofulven isadministered in a 3 week treatment regimen in which the irofulven isadministered on day 1 an day 8. The treatment regimen may be repeatedtwo to twenty times or more, as needed.

In particular, irofulven may be administered to the patient at a dose ofabout 0.05 mg/kg to 5 mg/kg, such as at a dose of about 0.1 mg/kg to 1mg/kg. For example, irofulven may be administered at a dose of about0.15 mg/kg, 0.20 mg/kg, 0.25 mg/kg, 0.30 mg/kg, 0.35 mg/kg, 0.40 mg/kg,0.45 mg/kg, 0.55 mg/kg, 0.60 mg/kg, 0.65 mg/kg, 0.70 mg/kg, 0.75 mg/kg,0.80 mg/kg, 0.85 mg/kg, 0.90 mg/kg, or 0.95 mg/kg. Irofulven may beadministered to the patient in a treatment regimen at least once perweek for at least two weeks and/or on day 1 and day 8 of a 3 weektreatment regimen, in which the treatment regimen occurs one or moretimes (e.g., the treatment regimen is repeated two to twenty times). Inparticular, irofulven is administered to the patient at a dose of about0.1 mg/kg to 1 mg/kg, such as a dose of about 0.2 mg/kg to about 0.6mg/kg (e.g., a dose of about 0.45 mg/kg). This dosage of irofulven canbe administered in a 3 week treatment regimen, in which irofulven isadministered on day 1 and day 8.

In the second or third aspect of the invention, the contacting step (a)and the measuring step (b) may occur prior to, concurrent with, or afteradministration of irofulven to the patient. Additionally, the contactingstep (a) and the measuring step (b) may occur two or more times, e.g.,during treatment with irofulven. For example, the contacting step (a)and the measuring step (b) may occur two or more times to assess thecontinued sensitivity of the patient to irofulven.

In any of the above aspects of the invention, the device can include atleast two, at least three, at least four, at least five, at least six,at least seven, at least eight, at least nine, at least ten, or moresingle-stranded nucleic acid molecules capable of specificallyhybridizing with the nucleotides of one or more biomarkers ofsensitivity selected from the biomarkers of Tables 1 and 3 (e.g., ATP1B1(SEQ ID NO: 201)); and/or at least two, at least three, at least four,at least five, at least six, at least seven, at least eight, at leastnine, at least ten, or more single-stranded nucleic acid moleculescapable of specifically hybridizing with the nucleotides of one or morebiomarkers of resistance selected from the biomarkers of Tables 2 and 4(e.g., IGLC1 (SEQ ID NO: 301, 302, 303, OR 318)). In particular, one ormore of the single-stranded nucleic acid molecules of the device have alength in the range of 10 to 100 nucleotides in length (e.g., .a lengthin the range of 20 to 60 nucleotides).

In any of the above aspects of the invention, the method may includeconverting the level of expression of one or more of the biomarkers ofsensitivity (e.g., one, two, three, four, five, ten, twenty, or all ofthe biomarkers shown in Tables 1 and 3, such as ATP1B1 (SEQ ID NO: 201))and/or one or more of the biomarkers of resistance (e.g., one, two,three, four, five, ten, twenty, or all of the biomarkers shown in Tables2 and 4, such as IGLC1 (SEQ ID NO: 301, 302, 303, OR 318)) into a meanscore, in which the mean score indicates the responsiveness of thepatient to irofulven. The method can further include subtracting themean score for one or more of the biomarkers of resistance (e.g., one,two, three, four, five, ten, twenty, or all of the biomarkers shown inTables 2 and 4, such as IGLC1 (SEQ ID NO: 301, 302, 303, OR 318)) fromthe mean score for one or more of the biomarkers of sensitivity (e.g.,one, two, three, four, five, ten, twenty, or all of the biomarkers shownin Tables 1 and 3, such as ATP1B1 (SEQ ID NO: 201)) to obtain adifference score, in which the difference score indicates theresponsiveness of the patient to irofulven. In particular, the meanscore and/or the difference score above a cutoff value indicates thatthe patient is responsive to irofulven, such as if the cutoff value isabout 0.1, about 0.15, about 0.2, about 0.25, about 0.3, about 0.35,about 0.4, about 0.45, about 0.5, or greater.

In any of the above aspects of the invention, the biomarker ofsensitivity may be selected from one or more of ATP1B1 (SEQ ID NO: 201or 219), UCHL1 (SEQ ID NO: 202), PTGR1 (SEQ ID NO: 203 or 210), NME7(SEQ ID NO: 204), PLS3 (SEQ ID NO: 205), S100A10 (SEQ ID NO: 206), CD24(SEQ ID NO: 207, 209, or 220), NQO1 (SEQ ID NO: 208 or 216), MYOF (SEQID NO: 211), LAPTM4B (SEQ ID NO: 212), CALD1 (SEQ ID NO: 213), PDGFC(SEQ ID NO: 214), BASP1 (SEQ ID NO: 215), ID1 (SEQ ID NO: 217), and GJA1(SEQ ID NO: 218). The biomarker of resistance may be selected from oneor more of IGLC1 (SEQ ID NO: 301, 302, 303, or 318), LAPTM5 (SEQ ID NO:304 or 338), ARHGDIB (SEQ ID NO: 305 or 311), SLC43A3 (SEQ ID NO: 307),LCP1 (SEQ ID NO: 308), HCLS1 (SEQ ID NO: 309), CD53 (SEQ ID NO: 310),MZB1 (SEQ ID NO: 313), RASSF5 (SEQ ID NO: 314 or 386), FAM46C (SEQ IDNO: 315), RCSD1 (SEQ ID NO: 316), IGJ (SEQ ID NO: 317), LPXN (SEQ ID NO:319), ITGB7 (SEQ ID NO: 320), and GTSF1 (SEQ ID NO: 321).

For example, the biomarkers of sensitivity may include ATP1B1 (SEQ IDNO: 201 or 219) and UCHL1 (SEQ ID NO: 202). The biomarkers ofsensitivity may include ATP1B1 (SEQ ID NO: 201 or 219), UCHL1 (SEQ IDNO: 202), and PTGR1 (SEQ ID NO: 203 or 210). The biomarkers ofsensitivity may include ATP1B1 (SEQ ID NO: 201 or 219), UCHL1 (SEQ IDNO: 202), PTGR1 (SEQ ID NO: 203 or 210), and NME7 (SEQ ID NO: 204). Thebiomarkers of sensitivity may include ATP1B1 (SEQ ID NO: 201 or 219),UCHL1 (SEQ ID NO: 202), PTGR1 (SEQ ID NO: 203 or 210), NME7 (SEQ ID NO:204), and PLS3 (SEQ ID NO: 205). The biomarkers of sensitivity mayinclude ATP1B1 (SEQ ID NO: 201 or 219), UCHL1 (SEQ ID NO: 202), PTGR1(SEQ ID NO: 203 or 210), NME7 (SEQ ID NO: 204), PLS3 (SEQ ID NO: 205),and S100A10 (SEQ ID NO: 206). The biomarkers of sensitivity may includeATP1B1 (SEQ ID NO: 201 or 219), UCHL1 (SEQ ID NO: 202), PTGR1 (SEQ IDNO: 203 or 210), NME7 (SEQ ID NO: 204), PLS3 (SEQ ID NO: 205), S100A10(SEQ ID NO: 206), and CD24 (SEQ ID NO: 207 or 209 or 220). Thebiomarkers of sensitivity may include ATP1B1 (SEQ ID NO: 201 or 219),UCHL1 (SEQ ID NO: 202), PTGR1 (SEQ ID NO: 203 or 210), NME7 (SEQ ID NO:204), PLS3 (SEQ ID NO: 205), S100A10 (SEQ ID NO: 206), CD24 (SEQ ID NO:207, 209, or 220), and NQO1 (SEQ ID NO: 208 or 216). The biomarkers ofsensitivity may include ATP1B1 (SEQ ID NO: 201 or 219), UCHL1 (SEQ IDNO: 202), PTGR1 (SEQ ID NO: 203 or 210), NME7 (SEQ ID NO: 204), PLS3(SEQ ID NO: 205), S100A10 (SEQ ID NO: 206), CD24 (SEQ ID NO: 207, 209,or 220), NQO1 (SEQ ID NO: 208 or 216), and MYOF (SEQ ID NO: 211). Thebiomarkers of sensitivity may include ATP1B1 (SEQ ID NO: 201 or 219),UCHL1 (SEQ ID NO: 202), PTGR1 (SEQ ID NO: 203 or 210), NME7 (SEQ ID NO:204), PLS3 (SEQ ID NO: 205), S100A10 (SEQ ID NO: 206), CD24 (SEQ ID NO:207, 209, or 220), NQO1 (SEQ ID NO: 208 or 216), MYOF (SEQ ID NO: 211),and LAPTM4B (SEQ ID NO: 212). The biomarkers of sensitivity may includeATP1B1 (SEQ ID NO: 201 or 219), UCHL1 (SEQ ID NO: 202), PTGR1 (SEQ IDNO: 203 or 210), NME7 (SEQ ID NO: 204), PLS3 (SEQ ID NO: 205), S100A10(SEQ ID NO: 206), CD24 (SEQ ID NO: 207, 209, or 220), NQO1 (SEQ ID NO:208 or 216), MYOF (SEQ ID NO: 211), LAPTM4B (SEQ ID NO: 212), and CALD1(SEQ ID NO: 213). The biomarkers of sensitivity may include ATP1B1 (SEQID NO: 201 or 219), UCHL1 (SEQ ID NO: 202), PTGR1 (SEQ ID NO: 203 or210), NME7 (SEQ ID NO: 204), PLS3 (SEQ ID NO: 205), S100A10 (SEQ ID NO:206), CD24 (SEQ ID NO: 207, 209, or 220), NQO1 (SEQ ID NO: 208 or 216),MYOF (SEQ ID NO: 211), LAPTM4B (SEQ ID NO: 212), CALD1 (SEQ ID NO: 213),and PDGFC (SEQ ID NO: 214). The biomarkers of sensitivity may includeATP1B1 (SEQ ID NO: 201 or 219), UCHL1 (SEQ ID NO: 202), PTGR1 (SEQ IDNO: 203 or 210), NME7 (SEQ ID NO: 204), PLS3 (SEQ ID NO: 205), S100A10(SEQ ID NO: 206), CD24 (SEQ ID NO: 207, 209, or 220), NQO1 (SEQ ID NO:208 or 216), MYOF (SEQ ID NO: 211), LAPTM4B (SEQ ID NO: 212), CALD1 (SEQID NO: 213), PDGFC (SEQ ID NO: 214), and BASP1 (SEQ ID NO: 215). Thebiomarkers of sensitivity may include ATP1B1 (SEQ ID NO: 201 or 219),UCHL1 (SEQ ID NO: 202), PTGR1 (SEQ ID NO: 203 or 210), NME7 (SEQ ID NO:204), PLS3 (SEQ ID NO: 205), S100A10 (SEQ ID NO: 206), CD24 (SEQ ID NO:207, 209, or 220), NQO1 (SEQ ID NO: 208 or 216), MYOF (SEQ ID NO: 211),LAPTM4B (SEQ ID NO: 212), CALD1 (SEQ ID NO: 213), PDGFC (SEQ ID NO:214), BASP1 (SEQ ID NO: 215), and ID1 (SEQ ID NO: 217). The biomarkersof sensitivity may include ATP1B1 (SEQ ID NO: 201 or 219), UCHL1 (SEQ IDNO: 202), PTGR1 (SEQ ID NO: 203 or 210), NME7 (SEQ ID NO: 204), PLS3(SEQ ID NO: 205), S100A10 (SEQ ID NO: 206), CD24 (SEQ ID NO: 207, 209,or 220), NQO1 (SEQ ID NO: 208 or 216), MYOF (SEQ ID NO: 211), LAPTM4B(SEQ ID NO: 212), CALD1 (SEQ ID NO: 213), PDGFC (SEQ ID NO: 214), BASP1(SEQ ID NO: 215), ID1 (SEQ ID NO: 217), and GJA1 (SEQ ID NO: 218).

For example, the biomarkers of resistance may include IGLC1 (SEQ ID NO:301, 302, 303, or 318) and LAPTM5 (SEQ ID NO: 304 or 338). Thebiomarkers of resistance may include IGLC1 (SEQ ID NO: 301, 302, 303, or318), LAPTM5 (SEQ ID NO: 304 or 338), and ARHGDIB (SEQ ID NO: 305 or311). The biomarkers of resistance may include IGLC1 (SEQ ID NO: 301,302, 303, or 318), LAPTM5 (SEQ ID NO: 304 or 338), ARHGDIB (SEQ ID NO:305 or 311), and SLC43A3 (SEQ ID NO: 307). The biomarkers of resistancemay include IGLC1 (SEQ ID NO: 301, 302, 303, or 318), LAPTM5 (SEQ ID NO:304 or 338), ARHGDIB (SEQ ID NO: 305 or 311), SLC43A3 (SEQ ID NO: 307),and LCP1 (SEQ ID NO: 308). The biomarkers of resistance may includeIGLC1 (SEQ ID NO: 301, 302, 303, or 318), LAPTM5 (SEQ ID NO: 304 or338), ARHGDIB (SEQ ID NO: 305 or 311), SLC43A3 (SEQ ID NO: 307), LCP1(SEQ ID NO: 308), and HCLS1 (SEQ ID NO: 309). The biomarkers ofresistance may include IGLC1 (SEQ ID NO: 301, 302, 303, or 318), LAPTM5(SEQ ID NO: 304 or 338), ARHGDIB (SEQ ID NO: 305 or 311), SLC43A3 (SEQID NO: 307), LCP1 (SEQ ID NO: 308), HCLS1 (SEQ ID NO: 309), and CD53(SEQ ID NO: 310). The IGLC1 (SEQ ID NO: 301, 302, 303, or 318), LAPTM5(SEQ ID NO: 304 or 338), ARHGDIB (SEQ ID NO: 305 or 311), SLC43A3 (SEQID NO: 307), LCP1 (SEQ ID NO: 308), HCLS1 (SEQ ID NO: 309), CD53 (SEQ IDNO: 310), and MZB1 (SEQ ID NO: 313). The biomarkers of resistance mayinclude IGLC1 (SEQ ID NO: 301, 302, 303, or 318), LAPTM5 (SEQ ID NO: 304or 338), ARHGDIB (SEQ ID NO: 305 or 311), SLC43A3 (SEQ ID NO: 307), LCP1(SEQ ID NO: 308), HCLS1 (SEQ ID NO: 309), CD53 (SEQ ID NO: 310), MZB1(SEQ ID NO: 313), and RASSF5 (SEQ ID NO: 314 or 386). The biomarkers ofresistance may include IGLC1 (SEQ ID NO: 301, 302, 303, or 318), LAPTM5(SEQ ID NO: 304 or 338), ARHGDIB (SEQ ID NO: 305 or 311), SLC43A3 (SEQID NO: 307), LCP1 (SEQ ID NO: 308), HCLS1 (SEQ ID NO: 309), CD53 (SEQ IDNO: 310), MZB1 (SEQ ID NO: 313), RASSF5 (SEQ ID NO: 314 or 386), andFAM46C (SEQ ID NO: 315). The biomarkers of resistance may include IGLC1(SEQ ID NO: 301, 302, 303, or 318), LAPTM5 (SEQ ID NO: 304 or 338),ARHGDIB (SEQ ID NO: 305 or 311), SLC43A3 (SEQ ID NO: 307), LCP1 (SEQ IDNO: 308), HCLS1 (SEQ ID NO: 309), CD53 (SEQ ID NO: 310), MZB1 (SEQ IDNO: 313), RASSF5 (SEQ ID NO: 314 or 386), FAM46C (SEQ ID NO: 315), andRCSD1 (SEQ ID NO: 316). The biomarkers of resistance may include IGLC1(SEQ ID NO: 301, 302, 303, or 318), LAPTM5 (SEQ ID NO: 304 or 338),ARHGDIB (SEQ ID NO: 305 or 311), SLC43A3 (SEQ ID NO: 307), LCP1 (SEQ IDNO: 308), HCLS1 (SEQ ID NO: 309), CD53 (SEQ ID NO: 310), MZB1 (SEQ IDNO: 313), RASSF5 (SEQ ID NO: 314 or 386), FAM46C (SEQ ID NO: 315), RCSD1(SEQ ID NO: 316), and IGJ (SEQ ID NO: 317). The biomarkers of resistancemay include IGLC1 (SEQ ID NO: 301, 302, 303, or 318), LAPTM5 (SEQ ID NO:304 or 338), ARHGDIB (SEQ ID NO: 305 or 311), SLC43A3 (SEQ ID NO: 307),LCP1 (SEQ ID NO: 308), HCLS1 (SEQ ID NO: 309), CD53 (SEQ ID NO: 310),MZB1 (SEQ ID NO: 313), RASSF5 (SEQ ID NO: 314 or 386), FAM46C (SEQ IDNO: 315), RCSD1 (SEQ ID NO: 316), IGJ (SEQ ID NO: 317), and LPXN (SEQ IDNO: 319). The biomarkers of resistance may include IGLC1 (SEQ ID NO:301, 302, 303, or 318), LAPTM5 (SEQ ID NO: 304 or 338), ARHGDIB (SEQ IDNO: 305 or 311), SLC43A3 (SEQ ID NO: 307), LCP1 (SEQ ID NO: 308), HCLS1(SEQ ID NO: 309), CD53 (SEQ ID NO: 310), MZB1 (SEQ ID NO: 313), RASSF5(SEQ ID NO: 314 or 386), FAM46C (SEQ ID NO: 315), RCSD1 (SEQ ID NO:316), IGJ (SEQ ID NO: 317), LPXN (SEQ ID NO: 319), and ITGB7 (SEQ ID NO:320). The biomarkers of resistance may include IGLC1 (SEQ ID NO: 301,302, 303, or 318), LAPTM5 (SEQ ID NO: 304 or 338), ARHGDIB (SEQ ID NO:305 or 311), SLC43A3 (SEQ ID NO: 307), LCP1 (SEQ ID NO: 308), HCLS1 (SEQID NO: 309), CD53 (SEQ ID NO: 310), MZB1 (SEQ ID NO: 313), RASSF5 (SEQID NO: 314 or 386), FAM46C (SEQ ID NO: 315), RCSD1 (SEQ ID NO: 316), IGJ(SEQ ID NO: 317), LPXN (SEQ ID NO: 319), ITGB7 (SEQ ID NO: 320), andGTSF1 (SEQ ID NO: 321).

In any of the above aspects of the invention, the device can be amicroarray, such as a deoxyribonucleic acid (DNA)-based platform.Alternatively, the device can be for performing a qRT-PCR reaction(e.g., the device is used with a system for detecting the amplificationproduct, for example, by fluorescence or by another method). The methodsmay also utilize both a microarray and a qRT-PCR. Thus, the expressionlevel of the biomarkers of sensitivity (e.g., one, two, three, four,five, ten, twenty, or all of the biomarkers shown in Tables 1 and 3,such as ATP1B1 (SEQ ID NO: 201)) and/or the biomarkers of resistance(e.g., one, two, three, four, five, ten, twenty, or all of thebiomarkers shown in Tables 2 and 4, such as IGLC1 (SEQ ID NO: 301, 302,303, OR 318)) can be measured using qRT-PCR. In particular, the level ofexpression of one or more of the biomarkers of sensitivity and/or one ormore of the biomarkers of resistance is determined by detecting thelevel of mRNA transcribed from one or more genes encoding one or more ofthe biomarkers of Tables 1-4.

In any of the above aspects of the invention, the cancer is selectedfrom a solid tumor cancer and a hematological cancer. For example, thecancer is prostate cancer, ovarian cancer, HCC, multiple myeloma, breastcancer, acute myelogenous leukemia (AML), acute lympho-blastic leukemia(ALL), chronic lymphocytic leukemia (CLL), myelodysplastic syndrome(MDS), chronic myelogenous leukemia-chronic phase (CMLCP), diffuse largeB-cell lymphoma (DLBCL), cutaneous T-cell lymphoma (CTCL), peripheralT-cell lymphoma (PTCL), Hodgkin's lymphoma, cervical cancer, renal cellcarcinoma (RCC), esophageal cancer, melanoma, glioma, pancreatic cancer,gastrointestinal stromal tumors (GIST), sarcoma, estrogenreceptor-positive (ERpos) breast cancer, non-small cell lung carcinoma(NSCLC), colon cancer, bladder cancer, or squamous cell carcinoma of thehead and neck (SCCHN). In particular, the cancer is prostate cancer,such as prostate cancer that is resistant to one or more cancertherapies other than irofulven. The cancer may also be ovarian cancer,such as ovarian cancer that is resistant to one or more cancer therapiesother than irofulven. Alternatively, the cancer may be HCC, such as HCCthat is resistant to one or more cancer therapies other than irofulven.

In any of the above aspects of the invention, the patient may exhibitcancer relapse (e.g., relapse of prostate cancer, ovarian cancer, orHCC), such as relapse after treatment with a therapeutic agent otherthan irofulven. In particular, the patient may exhibit cancer relapseprior to treatment with irofulven. Alternatively, the patient may havenot been administered a treatment for cancer.

Definitions

As used herein, “a” or “an” means “at least one” or “one or more” unlessotherwise indicated. In addition, the singular forms “a”, “an”, and“the” include plural referents unless the context clearly dictatesotherwise.

As used herein, “about” refers to an amount ±10% of the recited value.

By “biomarker” is meant a nucleic acid molecule (e.g., a mRNA or itscomplement, for example, a cDNA) or a protein encoded by the nucleicacid molecule present in, or from, a cell or tissue. The expression ofthe biomarker correlates to the responsiveness (e.g., sensitivity orresistance) of the cell or tissue (and thus, the patient in which thecell or tissue resides or the patient from which the cell or tissue wasobtained) to a cancer treatment (e.g., irofulven). In particular, abiomarker of sensitivity is a nucleic acid molecule (e.g., a mRNA or itscomplement) expressed from any one of the genes shown in Tables 1 and 3,or the protein encoded by the nucleic acid molecule, and a biomarker ofresistance is a nucleic acid molecule (e.g., a mRNA or its complement)expressed from any one of the genes shown in Tables 2 and 4, or theprotein encoded by the nucleic acid molecule.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals (e.g., humans) that is typicallycharacterized by unregulated cell proliferation. Examples of cancerinclude, but are not limited to, prostate cancer, ovarian cancer (e.g.,ovarian adenocarcinoma or embryonal carcinoma), liver cancer (e.g.,hepatocellular carcinoma (HCC) or hepatoma), myeloma (e.g., multiplemyeloma), colorectal cancer (e.g., colon cancer and rectal cancer),leukemia (e.g., acute myeloid leukemia, acute lymphoid leukemia, chronicmyeloid leukemia, chronic lymphocytic leukemia, acute myeloblasticleukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia,acute monocytic leukemia, acute erythroleukemia, and chronic leukemia),myelodysplastic syndrome, lymphoma (e.g., diffuse large B-cell lymphoma,cutaneous T-cell lymphoma, peripheral T-cell lymphoma, Hodgkin'slymphoma, non-Hodgkin's lymphoma, Waldenstrom's macroglobulinemia, andlymphocytic lymphoma), cervical cancer, esophageal cancer, melanoma,glioma (e.g., oligodendroglioma), pancreatic cancer (e.g., adenosquamouscarcinoma, signet ring cell carcinoma, hepatoid carcinoma, colloidcarcinoma, islet cell carcinoma, and pancreatic neuroendocrinecarcinoma), gastrointestinal stromal tumor, sarcoma (e.g., fibrosarcoma,myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma,angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, leiomyosarcoma, Ewing's sarcoma, andrhabdomyosarcoma), breast cancer (e.g., medullary carcinoma),ER-positive cancer, bladder cancer, head and neck cancer (e.g., squamouscell carcinoma of the head and neck), lung cancer (e.g., non-small celllung carcinoma, large cell carcinoma, bronchogenic carcinoma, andpapillary adenocarcinoma), metastatic cancer, oral cavity cancer,uterine cancer, testicular cancer (e.g., seminoma and embryonalcarcinoma), skin cancer (e.g., squamous cell carcinoma and basal cellcarcinoma), thyroid cancer (e.g., papillary carcinoma and medullarycarcinoma), brain cancer (e.g., astrocytoma and craniopharyngioma),stomach cancer, intra-epithelial cancer, bone cancer, biliary tractcancer, eye cancer, larynx cancer, kidney cancer (e.g., renal cellcarcinoma and Wilms tumor), gastric cancer, blastoma (e.g.,nephroblastoma, medulloblastoma, hemangioblastoma, neuroblastoma, andretinoblastoma), polycythemia vera, chordoma, synovioma, mesothelioma,adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma,cystadenocarcinoma, bile duct carcinoma, choriocarcinoma, epithelialcarcinoma, ependymoma, pinealoma, acoustic neuroma, schwannoma,meningioma, pituitary adenoma, nerve sheath tumor, cancer of the smallintestine, cancer of the endocrine system, cancer of the penis, cancerof the urethra, cutaneous or intraocular melanoma, a gynecologic tumor,solid tumors of childhood, and neoplasms of the central nervous system.The term cancer includes solid tumors (e.g., prostate cancer, ovariancancer, or hepatocellular carcinoma (HCC)) and hematological cancers(e.g., cancer of the blood, such as lymphoma (e.g., cutaneous T-celllymphoma (CTCL))).

The terms “expression level” and “level of expression,” as used herein,refer to the amount of a gene product in a cell, tissue, biologicalsample, organism, or patient, e.g., amounts of DNA, RNA (e.g. messengerRNA (mRNA)), or proteins corresponding to a given gene.

“Gene” as used herein indicates a coding or noncoding gene whoseactivity can be determined by measuring the produced RNA. Examplesinclude protein coding genes, microRNAs, small nuclear RNAs and otherRNAs with catalytic, regulatory or coding properties.

To “inhibit growth” as used herein means causing a reduction in cellgrowth (e.g., cancer cell growth, such as the NCI60 cancer cell lines)in vivo or in vitro by, e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 95%, or 99% or more, as evident by a reduction in the proliferationof cells exposed to a treatment (e.g., irofulven), relative to theproliferation of cells in the absence of the treatment. Growthinhibition may be the result of a treatment (e.g., irofulven) thatinduces apoptosis in a cell, induces necrosis in a cell, slows cellcycle progression, disrupts cellular metabolism, induces cell lysis, orinduces some other mechanism that reduces the proliferation of cells.

The term “irofulven” as used herein refers to an antitumor agent that isa cytotoxic semi-synthetic derivative of illudin S, a sesquiterpenetoxin found in the Jack ‘o’ Lantern mushroom. Irofulven or6-hydroxymethylacylfulvene is an alkylating agent that initiates theapoptotic degradation of DNA leading to cell death. Irofulven((6′R)-6′-hydroxy-3′-(hydroxymethyl)-2′,4′,6′-trimethylspiro[cyclopropane-1,5′-inden]-7′(6′H)-one)has the structural molecular formula shown below:

Irofulven is also described in MacDonald et al. (Cancer Res. 57 (2):279-283, 1997), hereby incorporated by reference.

“Microarray” as used herein means a device employed by any method thatquantifies one or more subject oligonucleotides, e.g., RNA, DNA, cDNA,or analogues thereof, at a time. For example, many DNA microarrays,including those made by Affymetrix (e.g., an Affymetrix HG-U133A orHG-U133_Plus_2 array), use several probes for determining the expressionof a single gene. The DNA microarray may contain oligonucleotide probesthat may be, e.g., full-length cDNAs complementary to an RNA or cDNAfragments that hybridize to part of an RNA. The DNA microarray may alsocontain modified versions of DNA or RNA, such as locked nucleic acids orLNA. Exemplary RNAs include mRNA, miRNA, and miRNA precursors.

As used herein, the term “percent (%) sequence identity” refers to thepercentage of nucleic acid residues of a candidate sequence, e.g., aprobe or primer of the invention, that are identical to the nucleic acidresidues of a reference sequence, e.g., a biomarker sequence of theinvention, after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent sequence identity (e.g., gapscan be introduced in one or both of the candidate and referencesequences for optimal alignment and non-homologous sequences can bedisregarded for comparison purposes). Alignment for purposes ofdetermining percent sequence identity can be achieved in various waysthat are within the skill in the art, for instance, using computersoftware, such as BLAST, BLAST-2, BLAST-P, BLAST-N, BLAST-X, WU-BLAST-2,ALIGN, ALIGN-2, CLUSTAL, Megalign (DNASTAR). In addition, those skilledin the art can determine appropriate parameters for measuring alignment,including any algorithms needed to achieve optimal alignment over thelength of the sequences being compared.

“NCI60” as used herein means a panel of 60 cancer cell lines from lung,colon, breast, ovarian, leukemia, renal, melanoma, prostate and braincancers including the following cancer cell lines: NSCLC_NCIH23,NSCLC_NCIH522, NSCLC_A549ATCC, NSCLC_EKVX, NSCLC_NCIH226,NSCLC_NCIH332M, NSCLC_H460, NSCLC_HOP62, NSCLC_HOP92, COLON_HT29,COLON_HCC-2998, COLON_HCT116, COLON_SW620, COLON_COLO205, COLON_HCT15,COLON_KM12, BREAST_MCF7, BREAST_MCF7ADRr, BREAST_MDAMB231,BREAST_HS578T, BREAST_MDAMB435, BREAST_MDN, BREAST_BT549, BREAST_T47D,OVAR_OVCAR3, OVAR_OVCAR4, OVAR_OVCAR5, OVAR_OVCAR8, OVAR_IGROV1,OVAR_SKOV3, LEUK_CCRFCEM, LEUK_K562, LEUK_MOLT4, LEUK_HL60,LEUK_RPMI8266, LEUK_SR, RENAL_UO31, RENAL_SN12C, RENAL_A498,RENAL_CAKI1, RENAL_RXF393, RENAL_7860, RENAL_ACHN, RENAL_TK10,MELAN_LOXIMVI, MELAN_MALME3M, MELAN_SKMEL2, MELAN_SKMEL5, MELAN_SKMEL28,MELAN_M14, MELAN_UACC62, MELAN_UACC257, PROSTATE_PC3, PROSTATE_DU145,CNS_SNB19, CNS_SNB75, CNS_U251, CNS_SF268, CNS_SF295, and CNS_SF539.

The terms “patient” and “subject,” as used interchangeably herein, referto any animal (e.g., a mammal, such as a human). A patient to be treatedor tested for responsiveness to a treatment (e.g., irofulven) accordingto the methods described herein may be one who has been diagnosed with acancer, such as those described herein, e.g., prostate cancer, ovariancancer, or hepatocellular carcinoma (HCC). Diagnosis may be performed byany method or techniques known in the art, such as x-ray, MRI, orbiopsy, and confirmed by a physician. To minimize exposure of a patientto drug treatments that may not be therapeutic, the patient may bedetermined to be either responsive or non-responsive to a cancertreatment, such as irofulven, according to the methods described herein.

“Resistant” or “resistance” as used herein means that a cell (e.g., acancer cell), a tissue (e.g., a tumor), or a patient having cancer(e.g., a human having cancer) is able to withstand treatment with ananti-cancer agent (e.g., irofulven). In particular, the treatment doesnot inhibit the growth of a cancer cell in vitro by about 70%, 80%, 90%,95%, 99% or 100% relative to the growth of a cancer cell not exposed tothe treatment. Resistance to treatment may be determined by a cellproliferation assay, e.g., a cell-based assay, which measures the growthof treated cells as a function of the absorbance of the cells of anincident light beam, such as the NCI60 assays described herein. In thisassay, greater absorbance indicates greater cell growth, and thus,resistance to the treatment.

A cancer patient (e.g., a patient with prostate cancer, ovarian cancer,or a hepatocellular carcinoma) may also have resistance to a cancertherapy other than irofulven, such as surgery, radiation, or atherapeutic agent (e.g., docetaxel, cabazitaxel, mitoxantrone,estramustine, prednisone, carboplatin, bevacizumab, paclitaxel,gemcitabine, doxorubicin, topotecan, etoposide, tamoxifen, letrozole,sorafenib, fluorouracil, capecitabine, oxaliplatin, interferon-alpha, or5-fluorouracil (5-FU)).

The terms “responsive” and “responsiveness,” as used herein, refer tothe likelihood that a cancer treatment (e.g., irofulven) has (e.g.,induces) a desired effect, or, alternatively, refer to the strength of adesired effect caused or induced by the treatment in a cell (e.g., acancer cell), a tissue (e.g., a tumor), or a patient having cancer(e.g., a human having cancer). For example, the desired effect caninclude inhibition of the growth of a cancer cell in vitro by more than10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% relative to thegrowth of a cancer cell not exposed to the treatment. The desired effectcan also include reduction in tumor mass by, e.g., about 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, or 100%. Responsiveness to treatment maybe determined by a cell proliferation assay, e.g., a cell-based assay,which measures the growth of treated cells as a function of theabsorbance of the cells of an incident light beam, such as the NCI60assays described herein. In this assay, lesser absorbance indicateslesser cell growth, and thus, sensitivity to the treatment. A greaterreduction in growth indicates more sensitivity to the treatment. Inparticular, “responsiveness” is a measure of the sensitivity orresistance of a patient to a treatment for cancer (e.g., irofulven).

The term “sample,” as used herein, refers to any specimen (such ascells, tissue (e.g., a tissue sample obtained by biopsy), blood, serum,plasma, urine, cerebrospinal fluid, or pancreatic fluid) taken from asubject. Preferably, the sample is taken from a portion of the bodyaffected by a cancer (e.g., a biopsy of the cancer tissue, such asprostate, ovarian, or lung cancer tissue). Biopsy may involve fineneedle aspiration biopsy, core needle biopsy (e.g., stereotactic coreneedle biopsy, vacuum-assisted core biopsy, or magnetic resonanceimaging (MRI) guided biopsy), or surgical biopsy (e.g., incisionalbiopsy or excisional biopsy). The sample may undergo additionalpurification and processing, for example, to remove cell debris andother unwanted molecules. Additional processing may further involveamplification, e.g., using PCR (RT-PCR). The standard methods of samplepurification, such as removal of unwanted molecules, are known in theart.

“Sensitive” and “sensitivity” as used herein refer to a cell (e.g., acancer cell), a tissue (e.g., a tumor), or a patient having cancer(e.g., a human having cancer) that is responsive to treatment, such asan anti-cancer agent (e.g., irofulven) or radiation treatment. Inparticular, the treatment inhibits the growth of a cancer cell in vitroby about 70%, 80%, 90%, 95%, 99% or 100% relative to the growth of acancer cell not exposed to the treatment. Sensitivity to treatment maybe determined by a cell proliferation assay, e.g., a cell-based assay,which measures the growth of treated cells as a function of theabsorbance of the cells of an incident light beam, such as the NCI60assays described herein. In this assay, lesser absorbance indicateslesser cell growth, and thus, sensitivity to the treatment.

“Treatment,” “medical treatment,” to “treat,” and “therapy,” as usedinterchangeably herein, refer to administering or exposing a patienthaving cancer (e.g., a human), a cell, or a tumor to an anti-canceragent (e.g., a drug, a protein, an antibody, a nucleic acid, achemotherapeutic agent, or a radioactive agent), or to some other formof medical intervention used to treat or prevent a disease, disorder, orcondition (e.g., surgery, cryotherapy, radiation therapy, orcombinations thereof). In particular, a medical treatment can includeirofulven. For example, the cancer to be treated is a solid tumor or ahematological cancer. Examples of cancer include, e.g., prostate cancer,ovarian cancer (e.g., ovarian adenocarcinoma or embryonal carcinoma),liver cancer (e.g., hepatocellular carcinoma (HCC) or hepatoma), myeloma(e.g., multiple myeloma), colorectal cancer (e.g., colon cancer andrectal cancer), leukemia (e.g., acute myeloid leukemia, acute lymphoidleukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, acutemyeloblastic leukemia, acute promyelocytic leukemia, acutemyelomonocytic leukemia, acute monocytic leukemia, acuteerythroleukemia, and chronic leukemia), myelodysplastic syndrome,lymphoma (e.g., diffuse large B-cell lymphoma, cutaneous T-celllymphoma, peripheral T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin'slymphoma, Waldenstrom's macroglobulinemia, and lymphocytic lymphoma),cervical cancer, esophageal cancer, melanoma, glioma (e.g.,oligodendroglioma), pancreatic cancer (e.g., adenosquamous carcinoma,signet ring cell carcinoma, hepatoid carcinoma, colloid carcinoma, isletcell carcinoma, and pancreatic neuroendocrine carcinoma),gastrointestinal stromal tumor, sarcoma (e.g., fibrosarcoma,myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma,angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, leiomyosarcoma, Ewing's sarcoma, andrhabdomyosarcoma), breast cancer (e.g., medullary carcinoma),ER-positive cancer, bladder cancer, head and neck cancer (e.g., squamouscell carcinoma of the head and neck), lung cancer (e.g., non-small celllung carcinoma, large cell carcinoma, bronchogenic carcinoma, andpapillary adenocarcinoma), metastatic cancer, oral cavity cancer,uterine cancer, testicular cancer (e.g., seminoma and embryonalcarcinoma), skin cancer (e.g., squamous cell carcinoma and basal cellcarcinoma), thyroid cancer (e.g., papillary carcinoma and medullarycarcinoma), brain cancer (e.g., astrocytoma and craniopharyngioma),stomach cancer, intra-epithelial cancer, bone cancer, biliary tractcancer, eye cancer, larynx cancer, kidney cancer (e.g., renal cellcarcinoma and Wilms tumor), gastric cancer, blastoma (e.g.,nephroblastoma, medulloblastoma, hemangioblastoma, neuroblastoma, andretinoblastoma), polycythemia vera, chordoma, synovioma, mesothelioma,adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma,cystadenocarcinoma, bile duct carcinoma, choriocarcinoma, epithelialcarcinoma, ependymoma, pinealoma, acoustic neuroma, schwannoma,meningioma, pituitary adenoma, nerve sheath tumor, cancer of the smallintestine, cancer of the endocrine system, cancer of the penis, cancerof the urethra, cutaneous or intraocular melanoma, a gynecologic tumor,solid tumors of childhood, and neoplasms of the central nervous system.Radiation therapy includes the administration of a radioactive agent toa patient or exposure of a patient to radiation. The radiation may begenerated from sources such as particle accelerators and related medicaldevices or agents that emit, e.g., X-radiation, gamma radiation, orelectron (Beta radiation) beams. A treatment may be or further includesurgery, e.g., to remove a tumor from a subject or living organism.

Other features and advantages of the invention will be apparent from thefollowing Detailed Description, the drawings, and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph grouping predicted sensitivity to irofulven by cancertype. Each gray circle represents the predicted irofulven sensitivity ofone patient calculated as the difference between the mean of theexpression levels of the biomarkers of sensitivity and the mean of theexpression levels of the biomarkers of resistance for the patient.Patients are grouped according to cancer type. The median predictedsensitivity (black bar) for a cancer type is related to the relativeresponse rate for that cancer type. The predictions are used forrelative comparisons to compare cancer types and cannot be used forabsolute predictions of response rate for a given cancer type. Thepredictions are normalized to a scale of 0 to 100 for all 3,522patients.

DETAILED DESCRIPTION OF THE INVENTION

I have discovered that the expression levels of the biomarkers shown inTables 1-4 may be detected in a patient having cancer and are useful forpredicting the responsiveness of the patient to irofulven. Thesepatients may already be determined to be resistance to a therapy otherthan irofulven, such as docetaxel, cabazitaxel, mitoxantrone,estramustine, prednisone, carboplatin, bevacizumab, paclitaxel,gemcitabine, doxorubicin, topotecan, etoposide, tamoxifen, letrozole,sorafenib, fluorouracil, capecitabine, oxaliplatin, interferon-alpha,5-fluorouracil (5-FU), a histone deacetylase (HDAC) inhibitor,ipilimumab, bortezomib, carfilzomib, thalidomide, lenalidomide,pomalidomide, dexamethasone, cyclophosphamide, vincristine, melphalan,tegafur, irinotecan, cetuximab, leucovorin, SN-38, everolimus,temsirolimus, bleomycin, lomustine, depsipeptide, erlotinib, cisplatin,busulfan, epirubicin, arsenic trioxide, bendamustine, fulvestrant,teniposide, adriamycin, decitabine, estramustine, azaguanine,aclarubicin, mitomycin, paclitaxel, taxotere, APO010, ara-c,methylprednisolone, methotrexate, methyl-gag, belinostat, idarubicin,IL4-PR38, valproic acid, all-trans retinoic acid (ATRA), cytoxan,suberoylanilide hydroxamic acid, leukeran, fludarabine, vinblastine,dacarbazine, hydroxyurea, tegafur, daunorubicin, mechlorethamine,streptozocin, carmustine, mercaptopurine, dactinomycin, tretinoin,ifosfamide, floxuridine, thioguanine, PSC 833, herceptin, celecoxib,iressa, anastrozole, or rituximab.

A device, such as a microarray, with one or more single-strandedoligonucleotide probes that have substantial identity (e.g., at least85%, 90%, 95%, 99%, or 100% sequence identity) to a sequence that iscomplementary or identical to the nucleic acid sequence of one or morebiomarkers shown in Tables 1-4 can be used according to the methodsdescribed herein to assess the responsiveness of a cancer patient totreatment with irofulven. For example, the probes can be used to detectone or more (e.g., two, three, four, five, ten, twenty, or all) of thebiomarkers of sensitivity listed in Tables 1 and 3, such as ATP1B1 (SEQID NO: 201 or 219), in a sample (e.g., a tumor sample) from a patienthaving cancer (e.g., prostate cancer, ovarian cancer, or hepatocellularcarcinoma (HCC)). Additionally, the probes can be used to detect one ormore (e.g., two, three, four, five, ten, twenty, or all) of thebiomarkers of resistance listed in Tables 2 and 4, such as IGLC1 (SEQ IDNO: 301, 302, 303, or 318), in a sample (e.g., a tumor sample) from apatient having cancer (e.g., prostate cancer, ovarian cancer, orhepatocellular carcinoma (HCC)).

Accordingly, the invention features individual biomarkers (e.g., ATP1B1(SEQ ID NO: 201 or 219) or IGLC1 (SEQ ID NO: 301, 302, 303, or 318)) andsets of biomarkers shown in Tables 1-4 that can be used to determine theresponsiveness of a cancer patient to irofulven at various stages ofdisease progression (e.g., patients diagnosed with cancer or patientsafter cancer recurrence) and at different times during the treatmentprocess (e.g., prior to administration of any cancer treatment, afteradministration of one or more cancer treatments other than irofulven,prior to administration of irofulven, or during administration ofirofulven). Additionally, the methods can be used to determine theirofulven responsiveness of a patient with cancer that is resistant toone or more cancer therapies other than irofulven, such as docetaxel,cabazitaxel, mitoxantrone, estramustine, prednisone, carboplatin,bevacizumab, paclitaxel, gemcitabine, doxorubicin, topotecan, etoposide,tamoxifen, letrozole, sorafenib, fluorouracil, capecitabine,oxaliplatin, interferon-alpha, 5-fluorouracil (5-FU), a histonedeacetylase (HDAC) inhibitor, ipilimumab, bortezomib, carfilzomib,thalidomide, lenalidomide, pomalidomide, dexamethasone,cyclophosphamide, vincristine, melphalan, tegafur, irinotecan,cetuximab, leucovorin, SN-38, everolimus, temsirolimus, bleomycin,lomustine, depsipeptide, erlotinib, cisplatin, busulfan, epirubicin,arsenic trioxide, bendamustine, fulvestrant, teniposide, adriamycin,decitabine, estramustine, azaguanine, aclarubicin, mitomycin,paclitaxel, taxotere, APO010, ara-c, methylprednisolone, methotrexate,methyl-gag, belinostat, idarubicin, IL4-PR38, valproic acid, all-transretinoic acid (ATRA), cytoxan, suberoylanilide hydroxamic acid,leukeran, fludarabine, vinblastine, dacarbazine, hydroxyurea, tegafur,daunorubicin, mechlorethamine, streptozocin, carmustine, mercaptopurine,dactinomycin, tretinoin, ifosfamide, floxuridine, thioguanine, PSC 833,herceptin, celecoxib, iressa, anastrozole, or rituximab.

In particular, the invention provides methods for determining whether apatient may be responsive to irofulven by, e.g., detecting theexpression level (e.g., mRNA or protein produced therefrom) of one ormore of the biomarkers shown in Tables 1-4 (e.g., ATP1B1 (SEQ ID NO: 201or 219)) in a biological sample (e.g., a tumor biopsy) obtained from thesubject using a device (e.g., a microarray or a protein array). Theexpression level of one or more of the biomarkers of sensitivity maythen be compared to the expression level of the biomarkers in a cell ortissue known to be sensitive or resistant to irofulven to determine thepatient's responsiveness to irofulven. The patient may be responsive toirofulven if the expression level of the one or more of the biomarkersof sensitivity (e.g., one or more of ATP1B1 (SEQ ID NO: 201 or 219),UCHL1 (SEQ ID NO: 202), PTGR1 (SEQ ID NO: 203 or 210), NME7 (SEQ ID NO:204), PLS3 (SEQ ID NO: 205), S100A10 (SEQ ID NO: 206), CD24 (SEQ ID NO:207, 209, or 220), NQO1 (SEQ ID NO: 208 or 216), MYOF (SEQ ID NO: 211),LAPTM4B (SEQ ID NO: 212), CALD1 (SEQ ID NO: 213), PDGFC (SEQ ID NO:214), BASP1 (SEQ ID NO: 215), ID1 (SEQ ID NO: 217), and GJA1 (SEQ ID NO:218)) is substantially similar to the expression level of the biomarkersof sensitivity in a cell or tissue known to be sensitive to irofulven(e.g., from a patient sensitive to irofulven). The patient may also beresponsive to irofulven if the level of expression of one or more of thebiomarkers of resistance (e.g., one or more of IGLC1 (SEQ ID NO: 301,302, 303, or 318), LAPTM5 (SEQ ID NO: 304 or 338), ARHGDIB (SEQ ID NO:305 or 311), SLC43A3 (SEQ ID NO: 307), LCP1 (SEQ ID NO: 308), HCLS1 (SEQID NO: 309), CD53 (SEQ ID NO: 310), MZB1 (SEQ ID NO: 313), RASSF5 (SEQID NO: 314 or 386), FAM46C (SEQ ID NO: 315), RCSD1 (SEQ ID NO: 316), IGJ(SEQ ID NO: 317), LPXN (SEQ ID NO: 319), ITGB7 (SEQ ID NO: 320), andGTSF1 (SEQ ID NO: 321)) is substantially dissimilar to the expressionlevel of the biomarkers of resistance in a cell or tissue known to beresistant to irofulven (e.g., from a patient resistant to irofulven).

The invention also features methods of treating a patient having cancer,such as a patient having a cancer that is resistant to one or morecancer therapies other than irofulven, by detecting the expressionlevels of one or more of the biomarkers shown in Tables 1-4 (e.g.,ATP1B1 (SEQ ID NO: 201)) in a sample (e.g., a tumor sample) from thepatient, and then administering irofulven based on the expression levelsof the biomarkers. In particular, a patient having cancer may beadministered irofulven if the expression level of one or more biomarkersof sensitivity is substantially similar to the expression level of thebiomarkers of sensitivity in a cell or tissue known to be sensitive toirofulven. Additionally, a patient having cancer may be administeredirofulven if the expression level of one or more biomarkers ofresistance is substantially dissimilar to the expression level of thebiomarkers of resistance in a cell or tissue known to be resistant toirofulven. Thus, the methods can be used to treat cancer patientspredicted to be responsive to irofulven, such as patients having, e.g.,prostate cancer, ovarian cancer, hepatocellular carcinoma (HCC),cervical cancer, renal cell carcinoma (RCC), esophageal cancer,melanoma, glioma, pancreatic cancer, gastrointestinal stromal tumors(GIST), sarcoma, estrogen receptor-positive (ERpos) breast cancer,non-small cell lung carcinoma (NSCLC), colon cancer, bladder cancer,squamous cell carcinoma of the head and neck (SCCHN), acute myelogenousleukemia (AML), acute lympho-blastic leukemia (ALL), chronic lymphocyticleukemia (CLL), myelodysplastic syndrome (MDS), chronic myelogenousleukemia-chronic phase (CMLCP), diffuse large B-cell lymphoma (DLBCL),cutaneous T-cell lymphoma (CTCL), peripheral T-cell lymphoma (PTCL), andHodgkin's lymphoma. Alternatively, a patient having cancer may not beadministered irofulven if the expression level of one or more biomarkersof sensitivity is substantially dissimilar to the expression level ofthe biomarkers of sensitivity in a cell or tissue known to be sensitiveto irofulven. Likewise, a patient having cancer may not be administeredirofulven if the expression level of one or more biomarkers ofresistance is substantially similar to the expression level of thebiomarkers of resistance in a cell or tissue known to be resistant toirofulven.

Methods are described herein for identifying biomarkers of drugresponsiveness, detecting biomarker gene expression in cancer patients,determining the responsiveness of a cancer patient to irofulven, andtreating cancer patients with irofulven. Also described are devices andkits for use in these methods.

Methods for Identifying Biomarkers of Drug Responsiveness

The invention features methods for identifying biomarkers (e.g., one ormore of the biomarkers of Tables 1-4) for determining the responsivenessof a cancer patient to a cancer treatment, such as irofulven. Suchmethods can involve, for example, an algorithm based on growthinhibition values (GI50) of cell lines (e.g., NCI60 cell lines)subjected to treatment with irofulven, followed by measurement of geneexpression (e.g., using a microarray (e.g., an Affymetrix HG-U133A orHG-U133_Plus_2 array)).

Methodology of the In Vitro Cancer Growth Inhibition Screen

The human tumor cell lines of the cancer screening panel may be grown inRPMI 1640 medium containing 5% fetal bovine serum and 2 mM L-glutamine.Cells may be inoculated into 96 well microtiter plates in 100 μL atplating densities ranging from 5,000 to 40,000 cells/well depending onthe doubling time of individual cell lines. After cell inoculation, themicrotiter plates may be incubated at 37° C., 5% CO2, 95% air and 100%relative humidity for 24 hours prior to addition of experimentalcompounds.

After 24 hours, two plates of each cell line may be fixed in situ withTCA, to represent a measurement of the cell population for each cellline at the time of compound addition (Tz). Experimental compounds maybe solubilized in dimethyl sulfoxide at 400-fold the desired finalmaximum test concentration and stored frozen prior to use. At the timeof compound (e.g., irofulven) addition, an aliquot of frozen concentratemay be thawed and diluted to twice the desired final maximum testconcentration with complete medium containing 50 μg/ml Gentamicin. Atotal of four additional 10-fold or ½ log serial dilutions are made toprovide a total of five concentrations plus control. Aliquots of 100 μlof these different compound dilutions are added to the appropriatemicrotiter wells already containing 100 μl of medium, resulting in therequired final compound concentrations.

Following compound (e.g., irofulven) addition, the plates may beincubated for an additional 48 h at 37° C., 5% CO2, 95% air, and 100%relative humidity. For adherent cells, the assay may be terminated bythe addition of cold TCA. Cells may be fixed in situ by the gentleaddition of 50 μl of cold 50% (w/v) TCA (final concentration, 10% TCA)and incubated for 60 minutes at 4° C. The supernatant may be discarded,and the plates may be washed five times with tap water and air-dried.Sulforhodamine B (SRB) solution (100 μl) at 0.4% (w/v) in 1% acetic acidmay be added to each well, and the plates may be incubated for 10minutes at room temperature. After staining, unbound dye may be removedby washing five times with 1% acetic acid and the plates may beair-dried. Bound stain may be subsequently solubilized with 10 mM trizmabase, and the absorbance may be read on an automated plate reader at awavelength of 515 nm. For suspension cells, the methodology may be thesame, except that the assay may be terminated by fixing settled cells atthe bottom of the wells by gently adding 50 μl of 80% TCA (finalconcentration, 16% TCA). Using the seven absorbance measurements [timezero, (Tz), control growth, (C), and test growth in the presence ofcompound (e.g., irofulven) at the five concentration levels (Ti)], thepercentage growth may be calculated at each of the compoundconcentrations levels.

Percentage growth inhibition may be calculated as:[(Ti−Tz)/(C−Tz)]×100 for concentrations for which Ti>/=Tz[(Ti−Tz)/Tz]×100 for concentrations for which Ti<Tz

Three dose response parameters may be calculated for each experimentalagent (e.g., irofulven). Growth inhibition of 50% (GI50) is calculatedfrom [(Ti−Tz)/(C−Tz)]×100=50, which is the agent (e.g., irofulven)concentration resulting in a 50% reduction in the net protein increase(as measured by SRB staining) in control cells during the compoundincubation. The compound concentration resulting in total growthinhibition (TGI) is calculated from Ti=Tz. The LC50 (concentration ofcompound resulting in a 50% reduction in the measured protein at the endof the compound treatment as compared to that at the beginning)indicating a net loss of cells following treatment is calculated from[(Ti−Tz)/Tz]×100=−50. Values are calculated for each of these threeparameters if the level of activity is reached; however, if the effectis not reached or is exceeded, the value for that parameter is expressedas greater or less than the maximum or minimum concentration tested.

Gene Expression and Growth Inhibition Analysis

The gene expression measurements of NCI60 cancer cell lines can beobtained from a publically available database (e.g., the National CancerInstitute and the Massachusetts Institute of Technology). Each datasetcan be normalized so that sample expression measured by different chipscan be compared. The preferred method of normalization is the logittransformation, which may be performed for each gene y on each chip, asfollows:logit(y)=log [(y−background)/(saturation−y)],

where background is calculated as the minimum intensity measured on thechip minus 0.1% of the signal intensity range: min−0.001*(max−min), andsaturation is calculated as the maximum intensity measured on the chipplus 0.1% of the signal intensity range: max+0.001*(max−min). Theresulting logit transformed data may then be z-transformed to mean zeroand standard deviation 1.

Next, gene expression can be correlated to cancer cell growthinhibition. Growth inhibition data (GI50) of the NCI60 cell lines in thepresence of a cancer treatment, such as irofulven, can be obtained fromthe NCI. The correlation between the logit-transformed expression levelof each gene in each cell line and the logarithm of GI50 (theconcentration of a given compound that results in a 50% inhibition ofgrowth) can be calculated, e.g., using the Pearson correlationcoefficient or the Spearman Rank-Order correlation coefficient. Insteadof using GI50s, any other measure of patient sensitivity to a giventreatment (e.g., irofulven) may be correlated to gene expression levelsof the patient. Since a plurality of measurements may be available for asingle gene, the most accurate determination of correlation coefficientcan be, e.g., the median of the correlation coefficients calculated forall probes measuring expression of the same gene.

For example, the median correlation coefficient of gene expressionmeasured on a probe to growth inhibition or patient sensitivity toirofulven can be calculated for all genes of interest. Genes that have amedian correlation above, e.g., 0.20, 0.21 0.22. 0.23, 0.24, 0.25, 0.26,0.27, 0.28, 0.29, 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38,0.39, 0.40, or higher (e.g., 0.2 or higher), can be used as biomarkersof sensitivity for assessing responsiveness of a cancer patient (e.g., apatient having cancer that is resistant to one or more cancer therapiesother than irofulven) to irofulven. Likewise, genes that have a mediancorrelation below, e.g., −0.20, −0.21, −0.22. −0.23, −0.24, −0.25,−0.26, −0.27, −0.28, −0.29, −0.30, −0.31, −0.32, −0.33, −0.34, −0.35,−0.36, −0.37, −0.38, −0.39, −0.40, or lower (e.g., −0.2 or lower), canbe used as biomarkers of resistance for assessing responsiveness of acancer patient (e.g., a patient having cancer that is resistant to oneor more cancer therapies other than irofulven) to irofulven. Preferably,the correlation coefficient of a biomarker of sensitivity will exceed0.2, while the correlation coefficient of a biomarker of resistance willbe less than −0.2. The result is a list of biomarker genes thatcorrelate to sensitivity or resistance to irofulven, as shown in Tables1 and 3 and Tables 2 and 4, respectively.

Cancer Types

The methods, devices, and kits of the invention can be used fordiagnosing, prognosing, monitoring, treating, and/or reducing cancer ina subject suffering from, diagnosed with, or susceptible to cancer.Non-limiting examples of cancers that can be diagnosed, prognosed,monitored, treated, or reduced using the methods include hematologicaland solid tumors. In particular, cancers include, e.g., prostate cancer,ovarian cancer (e.g., ovarian adenocarcinoma or embryonal carcinoma),liver cancer (e.g., hepatocellular carcinoma (HCC) or hepatoma), myeloma(e.g., multiple myeloma), colorectal cancer (e.g., colon cancer andrectal cancer), leukemia (e.g., acute myeloid leukemia, acute lymphoidleukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, acutemyeloblastic leukemia, acute promyelocytic leukemia, acutemyelomonocytic leukemia, acute monocytic leukemia, acuteerythroleukemia, and chronic leukemia), myelodysplastic syndrome,lymphoma (e.g., diffuse large B-cell lymphoma, cutaneous T-celllymphoma, peripheral T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin'slymphoma, Waldenstrom's macroglobulinemia, and lymphocytic lymphoma),cervical cancer, esophageal cancer, melanoma, glioma (e.g.,oligodendroglioma), pancreatic cancer (e.g., adenosquamous carcinoma,signet ring cell carcinoma, hepatoid carcinoma, colloid carcinoma, isletcell carcinoma, and pancreatic neuroendocrine carcinoma),gastrointestinal stromal tumor, sarcoma (e.g., fibrosarcoma,myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma,angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, leiomyosarcoma, Ewing's sarcoma, andrhabdomyosarcoma), breast cancer (e.g., medullary carcinoma),ER-positive cancer, bladder cancer, head and neck cancer (e.g., squamouscell carcinoma of the head and neck), lung cancer (e.g., non-small celllung carcinoma, large cell carcinoma, bronchogenic carcinoma, andpapillary adenocarcinoma), metastatic cancer, oral cavity cancer,uterine cancer, testicular cancer (e.g., seminoma and embryonalcarcinoma), skin cancer (e.g., squamous cell carcinoma and basal cellcarcinoma), thyroid cancer (e.g., papillary carcinoma and medullarycarcinoma), brain cancer (e.g., astrocytoma and craniopharyngioma),stomach cancer, intra-epithelial cancer, bone cancer, biliary tractcancer, eye cancer, larynx cancer, kidney cancer (e.g., renal cellcarcinoma and Wilms tumor), gastric cancer, blastoma (e.g.,nephroblastoma, medulloblastoma, hemangioblastoma, neuroblastoma, andretinoblastoma), polycythemia vera, chordoma, synovioma, mesothelioma,adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma,cystadenocarcinoma, bile duct carcinoma, choriocarcinoma, epithelialcarcinoma, ependymoma, pinealoma, acoustic neuroma, schwannoma,meningioma, pituitary adenoma, nerve sheath tumor, cancer of the smallintestine, cancer of the endocrine system, cancer of the penis, cancerof the urethra, cutaneous or intraocular melanoma, a gynecologic tumor,solid tumors of childhood, and neoplasms of the central nervous system.

In particular, the methods are useful for diagnosing, prognosing,monitoring, treating, or preventing, e.g., prostate cancer, ovariancancer, hepatocellular carcinoma (HCC), cervical cancer, renal cellcarcinoma (RCC), esophageal cancer, melanoma, glioma, pancreatic cancer,gastrointestinal stromal tumors (GIST), sarcoma, estrogenreceptor-positive (ERpos) breast cancer, non-small cell lung carcinoma(NSCLC), colon cancer, bladder cancer, squamous cell carcinoma of thehead and neck (SCCHN), acute myelogenous leukemia (AML), acutelympho-blastic leukemia (ALL), chronic lymphocytic leukemia (CLL),myelodysplastic syndrome (MDS), chronic myelogenous leukemia-chronicphase (CMLCP), diffuse large B-cell lymphoma (DLBCL), cutaneous T-celllymphoma (CTCL), peripheral T-cell lymphoma (PTCL), and Hodgkin'slymphoma.

For example, the cancer can be prostate cancer, such as Stage I, II(e.g., IIA or IIB), III, or IV prostate cancer. In particular, thecancer may be prostate cancer that is resistant to one or more cancertherapies, such as docetaxel, cabazitaxel, mitoxantrone, estramustine,prednisone, and/or surgery. Alternatively, the cancer is an ovariancancer. The ovarian cancer can be, for example, a Stage I (e.g., StageIA, IB, or IC), Stage II (e.g., Stage IIA or IIB), Stage III (e.g.,Stage IIIA1, IIIA2, IIIB, or IIIC), or Stage IV (e.g., Stage IVA or IVB)ovarian cancer. In particular, the cancer can be ovarian cancer that isresistant to one or more cancer therapies, such as docetaxel,carboplatin, bevacizumab, paclitaxel, gemcitabine, doxorubicin,topotecan, etoposide, tamoxifen, and/or letrozole. Additionally, thecancer can be HCC, such as Stage I, Stage II, Stage III (e.g., StageIIIA, IIIB, or IIIC), or Stage IV (e.g., Stage IVA or IVB) HCC. Inparticular, the cancer can be HCC that is resistant to one or morecancer therapies, such as sorafenib, doxorubicin, cisplatin,gemcitabine, capecitabine, oxaliplatin, interferon-alpha, and/or5-fluorouracil (5-FU).

Methods for Detecting Biomarker Gene Expression in Cancer Patients

A cancer patient can be assessed for sensitivity or resistance toirofulven by detecting gene expression of a biomarker (e.g., one or moreof the biomarkers of Tables 1-4) in a biological sample obtained fromthe cancer patient (e.g., a patient having cancer that is resistant toone or more cancer therapies other than irofulven). The biologicalsample can include, for example, cells, tissue (e.g., a tissue sampleobtained by biopsy), blood, serum, plasma, urine, sputum, cerebrospinalfluid, lymph tissue or fluid, or pancreatic fluid. For example, thebiological sample can be fresh frozen or formalin-fixed paraffinembedded (FFPE) tissue obtained from the subject, such as a tumor sample(e.g., a biopsy) from the tissue of interest (e.g., prostate, ovarian,lung, lymph nodes, thymus, spleen, bone marrow, breast, colorectal,pancreatic, cervical, bladder, gastrointestinal, head, or neck tissue).

RNA Extraction and Biomarker Expression Measurement

Cell samples or tissue samples may be snap frozen in liquid nitrogenuntil processing. RNA may be extracted using, e.g., Trizol Reagent fromInvitrogen following manufacturer's instructions, and detected directlyor converted to cDNA for detection. RNA may be amplified using, e.g.,MessageAmp kit from Ambion following manufacturer's instructions.Amplified RNA may be quantified using, e.g., HG-U133A or HG-U133_Plus2GeneChip from Affymetrix Inc. or a compatible apparatus, e.g., theGCS3000Dx GeneChip® System from Affymetrix Inc., using themanufacturer's instructions. The resulting biomarker expressionmeasurements may be further analyzed as described herein. The proceduresdescribed can be implemented using, e.g., R software available fromR-Project and supplemented with packages available from Bioconductor.

One or more of the biomarkers shown in Tables 1-4 (e.g., ATP1B1 (SEQ IDNO: 201)) may be measured in a biological sample (e.g., a tumor sample)obtained from the cancer patient (e.g., a patient with any of the cancertypes described herein, such as a patient having cancer that isresistant to one or more cancer therapies other than irofulven) using,e.g., polymerase chain reaction (PCR), reverse transcriptase PCR(RT-PCR), quantitative real-time PCR (qRT-PCR), an array (e.g., amicroarray), a genechip, pyrosequencing, nanopore sequencing, sequencingby synthesis, sequencing by expansion, single molecule real timetechnology, sequencing by ligation, microfluidics, infraredfluorescence, next generation sequencing (e.g., RNA-Seq techniques),Northern blots, Western blots, Southern blots, NanoString nCountertechnologies (e.g., those described in U.S. Patent Application Nos. US2011/0201515, US 2011/0229888, and US 2013/0017971, each of which isincorporated by reference in its entirety), proteomic techniques (e.g.,mass spectrometry or protein arrays), and combinations thereof.

Devices

Devices of the invention can be used for detecting the level ofexpression of one or more biomarkers shown in Tables 1-4. The device mayinclude at least one single-stranded nucleic acid (e.g., a probe) havingat least 85% sequence identity (e.g., 85%, 90%, 95%, 97%, 98%, 99%, or100% sequence identity) to a nucleic acid sequence that is complementaryor identical to at least 5 (e.g., at least 10, at least 15, at least 20,or more) consecutive nucleotides of one or more biomarkers shown inTables 1-4 (e.g., ATP1B1 (SEQ ID NO: 201 or 219) or IGLC1 (SEQ ID NO:301, 302, 303, or 318)), in which the at least one single-strandednucleic acid is sufficient for the detection of the expression level ofthe one or more biomarkers. The device may be used to detect theexpression level of a given biomarker by specific hybridization betweenthe single-stranded nucleic acid and the biomarker (e.g., an mRNA,genomic DNA, or non-coding RNA), a nucleic acid encoding the biomarker(e.g., an mRNA), or a complementary nucleic acid thereof. The device maybe or include a microarray. The device may also include or be used withreagents and materials for next generation sequence (e.g., sequencing bysynthesis). The device may also include or be used with NanoStringreagents and at least one nCounter cartridge. The device may be orinclude a protein array, which contains one or more protein bindingmoieties (e.g., proteins, antibodies, nucleic acids, aptamers,affibodies, lipids, phospholipids, small molecules, labeled variants ofany of the above, and any other moieties useful for protein detection aswell known in the art) capable of detectably binding to the polypeptideproduct(s) of one or more biomarkers shown in Tables 1-4. The device mayalso be a cartridge for measuring an amplification product resultingfrom hybridization between one or more nucleic acid molecules from thepatient and at least one single-stranded nucleic acid single-strandednucleic acid molecules of the device, such as a device for performingqRT-PCR.

Microarrays

The expression levels of the biomarkers (e.g., the biomarkers listed inTables 1-4 (e.g., ATP1B1 (SEQ ID NO: 201)) may be determined usinghigh-throughput expression profiling platforms, such as microarrays. Inparticular, a microarray for use in the methods for assessing theresponsiveness of a cancer patient (e.g., a patient having cancer thatis resistant to one or more cancer therapies other than irofulven) toirofulven contains or is produced by generating oligonucleotide probes(e.g., DNA, cDNA, or RNA probes) capable of hybridizing to one or morebiomarkers of interest (e.g., one or more of the biomarkers of Tables1-4) or the complement sequences thereof. Each probe can have, e.g., atleast 10, 15, 20, 25, 30, or more contiguous nucleic acid residues(e.g., at least 15) that are complementary or identical to a nucleicacid sequence of a selected biomarker. The probe nucleic sequence canalso have at least 85% (e.g., 90%, 95%, 99%, or 100%) sequence identityto the nucleic acid sequence of the gene coding the biomarker (e.g.,ATP1B1 (SEQ ID NO: 201 or 219)) or the complement sequence thereof. Inparticular, the probe sequences can be complementary to all or a portionof the nucleic acid sequence of the biomarker(s).

For example, microarrays of the invention for determining irofulvenresponsiveness can include probes for one or more (e.g., at least 5, 10,15, or 20 or more (e.g., all)) biomarkers of sensitivity shown in Tables1 and 3, such as ATP1B1 (SEQ ID NO: 201 or 219), UCHL1 (SEQ ID NO: 202),PTGR1 (SEQ ID NO: 203 or 210), NME7 (SEQ ID NO: 204), PLS3 (SEQ ID NO:205), S100A10 (SEQ ID NO: 206), CD24 (SEQ ID NO: 207, 209, or 220), NQO1(SEQ ID NO: 208 or 216), MYOF (SEQ ID NO: 211), LAPTM4B (SEQ ID NO:212), CALD1 (SEQ ID NO: 213), PDGFC (SEQ ID NO: 214), BASP1 (SEQ ID NO:215), ID1 (SEQ ID NO: 217), and/or GJA1 (SEQ ID NO: 218). Microarraysfor determining irofulven responsiveness can also include probes for oneor more (e.g., at least 5, 10, 15, or 20 or more (e.g., all)) biomarkersof resistance listed in Tables 2 and 4, such as IGLC1 (SEQ ID NO: 301,302, 303, or 318), LAPTM5 (SEQ ID NO: 304 or 338), ARHGDIB (SEQ ID NO:305 or 311), SLC43A3 (SEQ ID NO: 307), LCP1 (SEQ ID NO: 308), HCLS1 (SEQID NO: 309), CD53 (SEQ ID NO: 310), MZB1 (SEQ ID NO: 313), RASSF5 (SEQID NO: 314 or 386), FAM46C (SEQ ID NO: 315), RCSD1 (SEQ ID NO: 316), IGJ(SEQ ID NO: 317), LPXN (SEQ ID NO: 319), ITGB7 (SEQ ID NO: 320), and/orGTSF1 (SEQ ID NO: 321). Microarrays for determining irofulvenresponsiveness can also include probes for one or more (e.g., at least5, 10, 15, or 20 or more (e.g., all)) biomarkers of sensitivity andbiomarkers of resistance shown in Tables 1-4, such as ATP1B1 (SEQ ID NO:201 or 219), UCHL1 (SEQ ID NO: 202), PTGR1 (SEQ ID NO: 203 or 210), NME7(SEQ ID NO: 204), PLS3 (SEQ ID NO: 205), S100A10 (SEQ ID NO: 206), CD24(SEQ ID NO: 207, 209, or 220), NQO1 (SEQ ID NO: 208 or 216), MYOF (SEQID NO: 211), LAPTM4B (SEQ ID NO: 212), CALD1 (SEQ ID NO: 213), PDGFC(SEQ ID NO: 214), BASP1 (SEQ ID NO: 215), ID1 (SEQ ID NO: 217), GJA1(SEQ ID NO: 218), IGLC1 (SEQ ID NO: 301, 302, 303, or 318), LAPTM5 (SEQID NO: 304 or 338), ARHGDIB (SEQ ID NO: 305 or 311), SLC43A3 (SEQ ID NO:307), LCP1 (SEQ ID NO: 308), HCLS1 (SEQ ID NO: 309), CD53 (SEQ ID NO:310), MZB1 (SEQ ID NO: 313), RASSF5 (SEQ ID NO: 314 or 386), FAM46C (SEQID NO: 315), RCSD1 (SEQ ID NO: 316), IGJ (SEQ ID NO: 317), LPXN (SEQ IDNO: 319), ITGB7 (SEQ ID NO: 320), and/or GTSF1 (SEQ ID NO: 321).

A microarray probe may be single-stranded or double-stranded. The probemay be labeled (e.g., detectably labeled with a fluorescent molecule,dye molecule, small molecule, epitope tag, barcode sequence,polypeptide, or any other detectable molecule). Probes can be detectablylabeled and immobilized on a solid support to form the microarray. Forexample, probes can be either prefabricated and spotted to the surfaceor directly synthesized on to the surface (in situ) of the microarray.The microarray can also be configured such that the sequence andposition of each member (e.g., probe) of the array is known. Forexample, a selection of biomarkers whose expression correlates with anincreased likelihood of responsiveness to irofulven can be arrayed on asolid support. Hybridization of a labeled probe with a particular targetnucleic acid (e.g., an mRNA corresponding to one or more biomarkers ofTables 1-4) indicates that the sample from which the mRNA was derivedexpresses that biomarker (e.g., the biomarker of sensitivity orresistance to irofulven).

PCR-Based Techniques

As few as one to thirty (e.g., 5 to 30 or 10 to 30, or at least thefirst 15 of the biomarkers listed in Tables 1-2) biomarkers may be usedto determine patient responsiveness to irofulven using the methodsdescribed herein. Tissue or cell samples from a cancer patient (e.g., apatient having cancer that is resistant to one or more cancer therapiesother than irofulven) can be conveniently assayed for gene expressionlevels using polymerase chain reaction (PCR) analysis, such asquantitative real-time PCR (qPCR), or quantitative loop-mediatedisothermal amplification (q-LAMP). For example, an mRNA corresponding toa biomarker of Tables 1-4 can be detected in a biological sample by (a)producing cDNA from the sample by reverse transcription using at leastone primer; (b) amplifying the cDNA so produced using a targetpolynucleotide as sense and antisense primers to amplify target cDNAstherein; and (c) detecting the presence of the amplified target cDNAusing polynucleotide probes. The primers and probes including the targetsequences shown in Tables 1-4, such as ATP1B1 (SEQ ID NO: 201 or 219)and/or IGLC1 (SEQ ID NO: 301, 302, 303, or 318), may be used to detectexpression of one or more of the indicated biomarkers using PCR. Themethods can include one or more steps that allow determination of thelevels of target mRNA in a biological sample (e.g., by simultaneouslyexamining the levels of a comparative control mRNA sequence or“housekeeping” gene, such as an actin family member or GAPDH). Theprimers for these PCR-based assays may be labeled for detectionaccording to methods known in the art.

Sequencing

The expression levels of the biomarkers shown in Tables 1-4, such asATP1B1 (SEQ ID NO: 201 or 219) and/or IGLC1 (SEQ ID NO: 301, 302, 303,or 318), may be determined using sequencing technologies, such as nextgeneration sequencing platforms (e.g., RNA-Seq), as described inMortazavi et al., Nat. Methods 5: 621-628, 2008, hereby incorporated byreference. RNA-Seq is a robust technology for monitoring expression bydirect sequencing of the RNA molecules in a sample. This methodology mayinclude fragmentation of RNA to an average length of, e.g., 200nucleotides, conversion to cDNA by random priming, and synthesis ofdouble-stranded cDNA (e.g., using the PROTOSCRIPT® First Strand cDNASynthesis Kit from New England Biosciences). The cDNA may then beconverted into a molecular library for sequencing by addition ofsequence adapters for each library (e.g., from ILLUMINA®/Solexa), andthe resulting 50 to 100 nucleotide reads are mapped onto the genome.Exemplary sequencing platforms suitable for use according to the methodsinclude, e.g., pyrosequencing, ILLUMINA® sequencing by synthesis, SOLID®sequencing, ION TORRENT® sequencing, and SMRT® sequencing.

Methods of Determining the Responsiveness of a Patient to Irofulven

The invention features diagnostic methods for the detection andscreening of cancer patients (e.g., a patient having cancer that isresistant to one or more cancer therapies other than irofulven) that maybe responsive to irofulven using one or more of the biomarkers shown inTables 1-4 (e.g., ATP1B1 (SEQ ID NO: 201 or 219) or IGLC1 (SEQ ID NO:301, 302, 303, or 318)). The methods of the invention may be used forpredicting a patient's responsiveness to irofulven, and optionally,treating the cancer patient throughout the progression of cancer and/orin cases of recurrence (e.g., after a first line treatment, a secondline treatment, and/or a third line treatment).

The invention provides individual biomarkers (e.g., ATP1B1 (SEQ ID NO:201)) and sets of biomarkers (e.g., two or more of the biomarkers listedin Tables 1-4), the expression levels of which, as detected in abiological sample (e.g., a tumor sample, such as a biopsy) obtained froma cancer patient (e.g., a human with cancer), are indicative ofresponsiveness to irofulven. The biomarkers were identified usingmethods similar to those previously described in, e.g., Chen et al.(Mol. Cancer Ther. 11:34-33, 2012), Wang et al. (J. Nat. Cancer Inst.105: 1284-1291, 2013), and Knudsen et al. (PLoS One, 9: e87415, 2014),each of which are incorporated by reference herein in their entirety. Inparticular, an algorithm based on growth inhibition values (GI50) of acell line (e.g., NCI60 cells) is subjected to treatment with irofulvenand gene expression is determined (e.g., by microarray analysis, reversetranscriptase polymerase chain reaction (RT-PCR), quantitative real-timePCR (qPCR), or next generation sequencing). After normalization, geneswith, e.g., a Pearson correlation coefficient greater than about 0.2 orbelow about −0.2 can be classified as biomarkers of sensitivity orresistance, respectively. In particular, a correlation coefficient ofabout 0.2 or greater is a statistically significant cut-off known in theart for establishing whether the expression level of A GENE, e.g., thegenes shown in Tables 1-4, correlate with the likelihood of cancertreatment sensitivity, such as sensitivity to irofulven. Thus, acorrelation coefficient of about 0.2 or greater or about −0.2 or lowercan be used to estimate the statistical significance of the expressionlevel of the genes of Tables 1-4 for predicting patient responsivenessto treatment with irofulven according to the methods described herein.

Comparison of Biomarker Expression Levels

One or more biomarkers of sensitivity and/or resistance, identified asdescribed herein, can be used to predict responsiveness to irofulven bymeasuring the expression level of the biomarkers in a biological sampleobtained from the cancer patient. A single biomarker (e.g., any of thebiomarkers of Tables 1-4, such as ATP1B1 (SEQ ID NO: 201)) may be usedto determine the responsiveness of a cancer patient (e.g., a patienthaving cancer that is resistant to one or more cancer therapies otherthan irofulven) to irofulven. After determining the expression level ofthe biomarker(s) in a sample (e.g., a tumor sample) from the cancerpatient, the expression level of the biomarker(s) in the sample may becompared to the expression level of the biomarker(s) in a cell (e.g., acancer cell) or tissue (e.g., a tumor tissue) known to be sensitive totreatment with irofulven. If the expression level of the biomarker(s) inthe sample from the cancer patient is substantially similar (e.g.,identical to or has the same trend of expression level) to theexpression level of the biomarker(s) in the cell or tissue known to besensitive to irofulven, then the cancer patient is predicted to beresponsive to treatment with irofulven. Alternatively, if the expressionlevel of the biomarker(s) in the sample from the cancer patient issubstantially dissimilar to the expression level of the biomarker(s) inthe cell or tissue known to be sensitive to irofulven, then the cancerpatient is predicted to be non-responsive to treatment with irofulven.

The expression level of the biomarker (e.g., ATP1B1 (SEQ ID NO: 201)) ina sample from the cancer patient may also be compared to the expressionlevel of the biomarker in a cell (e.g., a cancer cell) or tissue (e.g.,a tumor tissue) known to be resistant to treatment with irofulven. Ifthe expression level of the biomarker in the sample from the cancerpatient is substantially similar to the expression level of thebiomarker in the cell or tissue known to be resistant to irofulven, thenthe cancer patient is predicted to be non-responsive to treatment withirofulven. Alternatively, if the expression level of the biomarker inthe sample from the cancer patient is substantially dissimilar to theexpression level of the biomarker in the cell or tissue known to besensitive to irofulven, then the cancer patient is predicted to beresponsive to treatment with irofulven.

The responsiveness of a cancer patient (e.g., a patient having cancerthat is resistant to one or more cancer therapies other than irofulven)to irofulven can also be predicted by comparing the expression level ofa biomarker (e.g., ATP1B1 (SEQ ID NO: 201)) to the expression level ofthe biomarker in one or more cells or tissues (e.g., from a cancerpatient population) known to be sensitive to treatment with irofulvenand one or more cells or tissues (e.g., from a cancer patientpopulation) known to be resistant to treatment with irofulven. Inparticular, the patient may be responsive to treatment with irofulven ifthe expression level of the biomarker is more similar to the expressionlevel of the biomarker in a cell or tissue known to be sensitive totreatment with irofulven than to a cell or tissue known to be resistantto treatment with irofulven. Alternatively, the patient may benon-responsive to treatment with irofulven if the expression level ofthe biomarker is more similar to the expression level of the biomarkerin a cell or tissue known to be resistant to treatment with irofulventhan to a cell or tissue known to be sensitive to treatment withirofulven.

Additionally, one or more biomarkers of sensitivity (e.g., one or moreof ATP1B1 (SEQ ID NO: 201 or 219), UCHL1 (SEQ ID NO: 202), PTGR1 (SEQ IDNO: 203 or 210), NME7 (SEQ ID NO: 204), PLS3 (SEQ ID NO: 205), S100A10(SEQ ID NO: 206), CD24 (SEQ ID NO: 207, 209, or 220), NQO1 (SEQ ID NO:208 or 216), MYOF (SEQ ID NO: 211), LAPTM4B (SEQ ID NO: 212), CALD1 (SEQID NO: 213), PDGFC (SEQ ID NO: 214), BASP1 (SEQ ID NO: 215), ID1 (SEQ IDNO: 217), and GJA1 (SEQ ID NO: 218)) and one or more biomarkers ofresistance (e.g., one or more of IGLC1 (SEQ ID NO: 301, 302, 303, or318), LAPTM5 (SEQ ID NO: 304 or 338), ARHGDIB (SEQ ID NO: 305 or 311),SLC43A3 (SEQ ID NO: 307), LCP1 (SEQ ID NO: 308), HCLS1 (SEQ ID NO: 309),CD53 (SEQ ID NO: 310), MZB1 (SEQ ID NO: 313), RASSF5 (SEQ ID NO: 314 or386), FAM46C (SEQ ID NO: 315), RCSD1 (SEQ ID NO: 316), IGJ (SEQ ID NO:317), LPXN (SEQ ID NO: 319), ITGB7 (SEQ ID NO: 320), and GTSF1 (SEQ IDNO: 321)) may be used in combination to determine the responsiveness ofa cancer patient (e.g., a patient having cancer that is resistant to oneor more cancer therapies other than irofulven) to treatment withirofulven. For example, the predicted responsiveness of a cancer patientmay be determined from, e.g., the difference score, which may be definedas the difference between the mean of the expression level of the one ormore biomarkers of sensitivity of Tables 1 and 3 and the mean of theexpression level of the one or more biomarkers of resistance of Tables 2and 4.

The difference score of the cancer patient can then be compared to thedifference score based on the expression level of the biomarkers in acell (e.g., a cancer cell) or tissue (e.g., a tumor tissue) known to besensitive or resistant to treatment with irofulven. In particular, thepatient may be responsive to treatment with irofulven if the differencescore is substantially similar to the expression level of the biomarkersin a cell or tissue known to be sensitive to treatment with irofulven.Alternatively, the patient may be non-responsive to treatment withirofulven if the difference score is substantially similar to theexpression level of the biomarkers in a cell or tissue known to beresistant to treatment with irofulven. Additionally, the patient may beresponsive to treatment with irofulven if the difference score issubstantially similar to the expression level of the biomarkers in acell or tissue known to be sensitive to treatment with irofulven than acell or tissue known to be resistant to treatment with irofulven.Alternatively, the patient may be non-responsive to treatment withirofulven if the difference score is substantially similar to theexpression level of the biomarkers in a cell or tissue known to beresistant to treatment with irofulven than a cell or tissue known to besensitive to treatment with irofulven.

One or more biomarkers of sensitivity and/or resistance, identified asdescribed herein, can be used to predict responsiveness to irofulven bymeasuring the expression level of the biomarkers in a biological sampleobtained from the cancer patient. A single biomarker (e.g., any of thebiomarkers of Tables 1-4, such as ATP1B1 (SEQ ID NO: 201)) may be usedto determine the responsiveness of a cancer patient (e.g., a patienthaving cancer that is resistant to one or more cancer therapies otherthan irofulven) to irofulven. After determining the expression level ofthe biomarker(s) in a sample (e.g., a tumor sample) from the cancerpatient, the expression level of the biomarker(s) in the sample may becompared to the expression level of the biomarker(s) in a cell (e.g., acancer cell) or tissue (e.g., a tumor tissue) known to be sensitive totreatment with irofulven. If the expression level of the biomarker(s) inthe sample from the cancer patient corresponds to (e.g., is identical toor has the same trend of expression level as) the expression level ofthe biomarker(s) in the cell or tissue known to be sensitive toirofulven, then the cancer patient is predicted to be responsive totreatment with irofulven. Alternatively, if the expression level of thebiomarker(s) in the sample from the cancer patient is substantiallydissimilar to the expression level of the biomarker(s) in the cell ortissue known to be sensitive to irofulven, then the cancer patient ispredicted to be non-responsive to treatment with irofulven.

The expression level of the biomarker (e.g., ATP1B1 (SEQ ID NO: 201)) ina sample from the cancer patient may also be compared to the expressionlevel of the biomarker in a cell (e.g., a cancer cell) or tissue (e.g.,a tumor tissue) known to be resistant to treatment with irofulven. Ifthe expression level of the biomarker in the sample from the cancerpatient corresponds to the expression level of the biomarker in the cellor tissue known to be resistant to irofulven, then the cancer patient ispredicted to be non-responsive to treatment with irofulven.Alternatively, if the expression level of the biomarker in the samplefrom the cancer patient is substantially dissimilar to the expressionlevel of the biomarker in the cell or tissue known to be resistant toirofulven, then the cancer patient is predicted to be responsive totreatment with irofulven.

The responsiveness of a cancer patient (e.g., a patient having cancerthat is resistant to one or more cancer therapies other than irofulven)to irofulven can also be predicted by comparing the expression level ofa biomarker (e.g., ATP1B1 (SEQ ID NO: 201)) to the expression level ofthe biomarker in one or more cells or tissues (e.g., from a cancerpatient population) known to be sensitive to treatment with irofulvenand one or more cells or tissues (e.g., from a cancer patientpopulation) known to be resistant to treatment with irofulven. Inparticular, the patient may be responsive to treatment with irofulven ifthe expression level of the biomarker(s) corresponds to the expressionlevel of the biomarker(s) in a cell or tissue known to be sensitive totreatment with irofulven relative to the expression level of thebiomarkers in a cell or tissue known to be resistant to treatment withirofulven. Alternatively, the patient may be non-responsive to treatmentwith irofulven if the expression level of the biomarker(s) correspondsto the expression level of the biomarker(s) in a cell or tissue known tobe resistant to treatment with irofulven relative to the expressionlevel of the biomarkers in a cell or tissue known to be resistant totreatment with irofulven.

Additionally, one or more biomarkers of sensitivity (e.g., one or moreof ATP1B1 (SEQ ID NO: 201 or 219), UCHL1 (SEQ ID NO: 202), PTGR1 (SEQ IDNO: 203 or 210), NME7 (SEQ ID NO: 204), PLS3 (SEQ ID NO: 205), S100A10(SEQ ID NO: 206), CD24 (SEQ ID NO: 207, 209, or 220), NQO1 (SEQ ID NO:208 or 216), MYOF (SEQ ID NO: 211), LAPTM4B (SEQ ID NO: 212), CALD1 (SEQID NO: 213), PDGFC (SEQ ID NO: 214), BASP1 (SEQ ID NO: 215), ID1 (SEQ IDNO: 217), and GJA1 (SEQ ID NO: 218)) and one or more biomarkers ofresistance (e.g., one or more of IGLC1 (SEQ ID NO: 301, 302, 303, or318), LAPTM5 (SEQ ID NO: 304 or 338), ARHGDIB (SEQ ID NO: 305 or 311),SLC43A3 (SEQ ID NO: 307), LCP1 (SEQ ID NO: 308), HCLS1 (SEQ ID NO: 309),CD53 (SEQ ID NO: 310), MZB1 (SEQ ID NO: 313), RASSF5 (SEQ ID NO: 314 or386), FAM46C (SEQ ID NO: 315), RCSD1 (SEQ ID NO: 316), IGJ (SEQ ID NO:317), LPXN (SEQ ID NO: 319), ITGB7 (SEQ ID NO: 320), and GTSF1 (SEQ IDNO: 321)) may be used in combination to determine the responsiveness ofa cancer patient (e.g., a patient having cancer that is resistant to oneor more cancer therapies other than irofulven) to treatment withirofulven. For example, the predicted responsiveness of a cancer patientmay be determined from, e.g., the difference score, which may be definedas the difference between the mean of the expression level of the one ormore biomarkers of sensitivity of Tables 1 and 3 and the mean of theexpression level of the one or more biomarkers of resistance of Tables 2and 4.

The difference score of the cancer patient can then be compared to thedifference score based on the expression level of the biomarkers in acell (e.g., a cancer cell) or tissue (e.g., a tumor tissue) known to besensitive or resistant to treatment with irofulven. In particular, thepatient may be responsive to treatment with irofulven if the differencescore corresponds to the expression level of the biomarkers in a cell ortissue known to be sensitive to treatment with irofulven. Alternatively,the patient may be non-responsive to treatment with irofulven if thedifference score corresponds to the expression level of the biomarkersin a cell or tissue known to be resistant to treatment with irofulven.Additionally, the patient may be responsive to treatment with irofulvenif the difference score corresponds to the expression level of thebiomarkers in a cell or tissue known to be sensitive to treatment withirofulven relative to the expression level of the biomarkers in a cellor tissue known to be resistant to treatment with irofulven.Alternatively, the patient may be non-responsive to treatment withirofulven if the difference score corresponds to the expression level ofthe biomarkers in a cell or tissue known to be resistant to treatmentwith irofulven relative to the expression level of the biomarkers in acell or tissue known to be sensitive to treatment with irofulven.

Preferably, the cell or tissue known to be either sensitive or resistantto irofulven is of the same cancer type as the cancer patient with anunknown responsiveness to irofulven. For example, the cancer patient andthe cell or tissue known to be either sensitive or resistant toirofulven may both have a cancer type selected from a solid tumor or ahematological cancer, e.g., prostate cancer, ovarian cancer (e.g.,ovarian adenocarcinoma or embryonal carcinoma), liver cancer (e.g.,hepatocellular carcinoma (HCC) or hepatoma), myeloma (e.g., multiplemyeloma), colorectal cancer (e.g., colon cancer and rectal cancer),leukemia (e.g., acute myeloid leukemia, acute lymphoid leukemia, chronicmyeloid leukemia, chronic lymphocytic leukemia, acute myeloblasticleukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia,acute monocytic leukemia, acute erythroleukemia, and chronic leukemia),myelodysplastic syndrome, lymphoma (e.g., diffuse large B-cell lymphoma,cutaneous T-cell lymphoma, peripheral T-cell lymphoma, Hodgkin'slymphoma, non-Hodgkin's lymphoma, Waldenstrom's macroglobulinemia, andlymphocytic lymphoma), cervical cancer, esophageal cancer, melanoma,glioma (e.g., oligodendroglioma), pancreatic cancer (e.g., adenosquamouscarcinoma, signet ring cell carcinoma, hepatoid carcinoma, colloidcarcinoma, islet cell carcinoma, and pancreatic neuroendocrinecarcinoma), gastrointestinal stromal tumor, sarcoma (e.g., fibrosarcoma,myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma,angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, leiomyosarcoma, Ewing's sarcoma, andrhabdomyosarcoma), breast cancer (e.g., medullary carcinoma),ER-positive cancer, bladder cancer, head and neck cancer (e.g., squamouscell carcinoma of the head and neck), lung cancer (e.g., non-small celllung carcinoma, large cell carcinoma, bronchogenic carcinoma, andpapillary adenocarcinoma), metastatic cancer, oral cavity cancer,uterine cancer, testicular cancer (e.g., seminoma and embryonalcarcinoma), skin cancer (e.g., squamous cell carcinoma and basal cellcarcinoma), thyroid cancer (e.g., papillary carcinoma and medullarycarcinoma), brain cancer (e.g., astrocytoma and craniopharyngioma),stomach cancer, intra-epithelial cancer, bone cancer, biliary tractcancer, eye cancer, larynx cancer, kidney cancer (e.g., renal cellcarcinoma and Wilms tumor), gastric cancer, blastoma (e.g.,nephroblastoma, medulloblastoma, hemangioblastoma, neuroblastoma, andretinoblastoma), polycythemia vera, chordoma, synovioma, mesothelioma,adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma,cystadenocarcinoma, bile duct carcinoma, choriocarcinoma, epithelialcarcinoma, ependymoma, pinealoma, acoustic neuroma, schwannoma,meningioma, pituitary adenoma, nerve sheath tumor, cancer of the smallintestine, cancer of the endocrine system, cancer of the penis, cancerof the urethra, cutaneous or intraocular melanoma, a gynecologic tumor,solid tumors of childhood, and neoplasms of the central nervous system.In particular, the cancer of the patient and the cell or tissue withknown resistance or sensitivity to irofulven is, e.g., prostate cancer,ovarian cancer, hepatocellular carcinoma (HCC), multiple myeloma, breastcancer, acute myelogenous leukemia (AML), acute lympho-blastic leukemia(ALL), chronic lymphocytic leukemia (CLL), myelodysplastic syndrome(MDS), chronic myelogenous leukemia-chronic phase (CMLCP), diffuse largeB-cell lymphoma (DLBCL), cutaneous T-cell lymphoma (CTCL), peripheralT-cell lymphoma (PTCL), Hodgkin's lymphoma, cervical cancer, renal cellcarcinoma (RCC), esophageal cancer, melanoma, glioma, pancreatic cancer,gastrointestinal stromal tumors (GIST), sarcoma, estrogenreceptor-positive (ERpos) breast cancer, non-small cell lung carcinoma(NSCLC), colon cancer, bladder cancer, or squamous cell carcinoma of thehead and neck (SCCHN).

Machine learning techniques such as Neural Networks, Support VectorMachines, K Nearest Neighbor, and Nearest Centroids may also be employedto develop models that discriminate patients sensitive to treatment withirofulven from those resistant to treatment with irofulven usingbiomarker expression as model variables which assign each patient aclassification as sensitive or resistant to treatment with irofulven.Machine learning techniques used to classify patients using variousmeasurements are described in U.S. Pat. No. 5,822,715; U.S. PatentApplication Publication Nos. 2003/0073083, 2005/0227266, 2005/0208512,2005/0123945, 2003/0129629, and 2002/0006613; and in Vapnik V N.Statistical Learning Theory, John Wiley & Sons, New York, 1998; Hastieet al., 2001, The Elements of Statistical Learning: Data Mining,Inference, and Prediction, Springer, N.Y.; Agresti, 1996, AnIntroduction to Categorical Data Analysis, John Wiley & Sons, New York;V. Tresp et al., “Neural Network Modeling of Physiological Processes,”in Hanson S. J. et al. (Eds.), Computational Learning Theory and NaturalLearning Systems 2, MIT Press, 1994, each of which are herebyincorporated by reference in their entirety.

Biomarkers of Sensitivity and Resistance

The expression levels of one or more biomarkers of Tables 1-4 can beused to determine cancer patient responsiveness to treatment withirofulven. Once determined to be responsive, the patient can be treatedwith irofulven. In particular, the biomarker ATP1B1 (SEQ ID NO: 201 or219) may be used to assess a cancer patient's (e.g, a patient havingcancer that is resistant to one or more cancer therapies other thanirofulven) responsiveness to irofulven. The expression level of thebiomarker ATP1B1 (SEQ ID NO: 201 or 219) may be assessed using nucleicacid amplification methods (e.g., PCR) or a device (e.g., a microarray).As is described above, the expression level of ATP1B1 (SEQ ID NO: 201 or219) in the patient sample may then be compared, e.g., to the expressionlevel of ATP1B1 (SEQ ID NO: 201 or 219) in a cell (e.g., a cancer cell)or tissue (e.g., a tumor tissue) known to be sensitive or resistant totreatment with irofulven and used to determine the cancer patient'sresponsiveness to irofulven. The biomarker ATP1B1 (SEQ ID NO: 201 or219) may be used alone to predict cancer patient responsiveness totreatment with irofulven or in combination with one or more additionalbiomarkers (e.g., one, two, three, four, five, ten, or all of thebiomarkers shown in Tables 1-4), such as UCHL1 (SEQ ID NO: 202), PTGR1(SEQ ID NO: 203 or 210), NME7 (SEQ ID NO: 204), PLS3 (SEQ ID NO: 205),S100A10 (SEQ ID NO: 206), CD24 (SEQ ID NO: 207, 209, or 220), NQO1 (SEQID NO: 208 or 216), MYOF (SEQ ID NO: 211), LAPTM4B (SEQ ID NO: 212),CALD1 (SEQ ID NO: 213), PDGFC (SEQ ID NO: 214), BASP1 (SEQ ID NO: 215),ID1 (SEQ ID NO: 217), GJA1 (SEQ ID NO: 218), IGLC1 (SEQ ID NO: 301, 302,303, or 318), LAPTM5 (SEQ ID NO: 304 or 338), ARHGDIB (SEQ ID NO: 305 or311), SLC43A3 (SEQ ID NO: 307), LCP1 (SEQ ID NO: 308), HCLS1 (SEQ ID NO:309), CD53 (SEQ ID NO: 310), MZB1 (SEQ ID NO: 313), RASSF5 (SEQ ID NO:314 or 386), FAM46C (SEQ ID NO: 315), RCSD1 (SEQ ID NO: 316), IGJ (SEQID NO: 317), LPXN (SEQ ID NO: 319), ITGB7 (SEQ ID NO: 320), and/or GTSF1(SEQ ID NO: 321). The expression level of the biomarker(s) may bedetermined using, e.g., a microarray, PCR, or other techniques describedherein, for example, using a nucleic acid probe sequence based on thetarget sequences shown in Tables 1-4.

The expression level of the biomarker UCHL1 (SEQ ID NO: 202) may beassessed using nucleic acid amplification methods (e.g., PCR) or adevice (e.g., a microarray). As is described above, the expression levelof UCHL1 (SEQ ID NO: 202) in the patient sample may then be compared,e.g., to the expression level of UCHL1 (SEQ ID NO: 202) in a cell (e.g.,a cancer cell) or tissue (e.g., a tumor tissue) known to be sensitive orresistant to treatment with irofulven and used to determine the cancerpatient's responsiveness to irofulven. The biomarker UCHL1 (SEQ ID NO:202) may be used alone to predict cancer patient responsiveness totreatment with irofulven or in combination with one or more additionalbiomarkers (e.g., one, two, three, four, five, ten, or all of thebiomarkers shown in Tables 1-4), such as ATP1B1 (SEQ ID NO: 201 or 219),PTGR1 (SEQ ID NO: 203 or 210), NME7 (SEQ ID NO: 204), PLS3 (SEQ ID NO:205), S100A10 (SEQ ID NO: 206), CD24 (SEQ ID NO: 207, 209, or 220), NQO1(SEQ ID NO: 208 or 216), MYOF (SEQ ID NO: 211), LAPTM4B (SEQ ID NO:212), CALD1 (SEQ ID NO: 213), PDGFC (SEQ ID NO: 214), BASP1 (SEQ ID NO:215), ID1 (SEQ ID NO: 217), GJA1 (SEQ ID NO: 218), IGLC1 (SEQ ID NO:301, 302, 303, or 318), LAPTM5 (SEQ ID NO: 304 or 338), ARHGDIB (SEQ IDNO: 305 or 311), SLC43A3 (SEQ ID NO: 307), LCP1 (SEQ ID NO: 308), HCLS1(SEQ ID NO: 309), CD53 (SEQ ID NO: 310), MZB1 (SEQ ID NO: 313), RASSF5(SEQ ID NO: 314 or 386), FAM46C (SEQ ID NO: 315), RCSD1 (SEQ ID NO:316), IGJ (SEQ ID NO: 317), LPXN (SEQ ID NO: 319), ITGB7 (SEQ ID NO:320), and/or GTSF1 (SEQ ID NO: 321). The expression level of thebiomarker(s) may be determined using, e.g., a microarray, PCR, or othertechniques described herein, for example, using a nucleic acid probesequence based on the target sequences shown in Tables 1-4.

The biomarker PTGR1 (SEQ ID NO: 203 or 210) may be used to assess acancer patient's (e.g, a patient having cancer that is resistant to oneor more cancer therapies other than irofulven) responsiveness toirofulven. The expression level of the biomarker PTGR1 (SEQ ID NO: 203or 210) may be assessed using nucleic acid amplification methods (e.g.,PCR) or a device (e.g., a microarray). As is described above, theexpression level of PTGR1 (SEQ ID NO: 203 or 210) in the patient samplemay then be compared, e.g., to the expression level of PTGR1 (SEQ ID NO:203 or 210) in a cell (e.g., a cancer cell) or tissue (e.g., a tumortissue) known to be sensitive or resistant to treatment with irofulvenand used to determine the cancer patient's responsiveness to irofulven.The biomarker PTGR1 (SEQ ID NO: 203 or 210) may be used alone to predictcancer patient responsiveness to treatment with irofulven or incombination with one or more additional biomarkers (e.g., one, two,three, four, five, ten, or all of the biomarkers shown in Tables 1-4),such as ATP1B1 (SEQ ID NO: 201 or 219), UCHL1 (SEQ ID NO: 202), NME7(SEQ ID NO: 204), PLS3 (SEQ ID NO: 205), S100A10 (SEQ ID NO: 206), CD24(SEQ ID NO: 207, 209, or 220), NQO1 (SEQ ID NO: 208 or 216), MYOF (SEQID NO: 211), LAPTM4B (SEQ ID NO: 212), CALD1 (SEQ ID NO: 213), PDGFC(SEQ ID NO: 214), BASP1 (SEQ ID NO: 215), ID1 (SEQ ID NO: 217), GJA1(SEQ ID NO: 218), IGLC1 (SEQ ID NO: 301, 302, 303, or 318), LAPTM5 (SEQID NO: 304 or 338), ARHGDIB (SEQ ID NO: 305 or 311), SLC43A3 (SEQ ID NO:307), LCP1 (SEQ ID NO: 308), HCLS1 (SEQ ID NO: 309), CD53 (SEQ ID NO:310), MZB1 (SEQ ID NO: 313), RASSF5 (SEQ ID NO: 314 or 386), FAM46C (SEQID NO: 315), RCSD1 (SEQ ID NO: 316), IGJ (SEQ ID NO: 317), LPXN (SEQ IDNO: 319), ITGB7 (SEQ ID NO: 320), and/or GTSF1 (SEQ ID NO: 321). Theexpression level of the biomarker(s) may be determined using, e.g., amicroarray, PCR, or other techniques described herein, for example,using a nucleic acid probe sequence based on the target sequences shownin Tables 1-4.

The biomarker NME7 (SEQ ID NO: 204) may be used to assess a cancerpatient's (e.g, a patient having cancer that is resistant to one or morecancer therapies other than irofulven) responsiveness to irofulven. Theexpression level of the biomarker NME7 (SEQ ID NO: 204) may be assessedusing nucleic acid amplification methods (e.g., PCR) or a device (e.g.,a microarray). As is described above, the expression level of NME7 (SEQID NO: 204) in the patient sample may then be compared, e.g., to theexpression level of NME7 (SEQ ID NO: 204) in a cell (e.g., a cancercell) or tissue (e.g., a tumor tissue) known to be sensitive orresistant to treatment with irofulven and used to determine the cancerpatient's responsiveness to irofulven. The biomarker NME7 (SEQ ID NO:204) may be used alone to predict cancer patient responsiveness totreatment with irofulven or in combination with one or more additionalbiomarkers (e.g., one, two, three, four, five, ten, or all of thebiomarkers shown in Tables 1-4), such as ATP1B1 (SEQ ID NO: 201 or 219),UCHL1 (SEQ ID NO: 202), PTGR1 (SEQ ID NO: 203 or 210), PLS3 (SEQ ID NO:205), S100A10 (SEQ ID NO: 206), CD24 (SEQ ID NO: 207, 209, or 220), NQO1(SEQ ID NO: 208 or 216), MYOF (SEQ ID NO: 211), LAPTM4B (SEQ ID NO:212), CALD1 (SEQ ID NO: 213), PDGFC (SEQ ID NO: 214), BASP1 (SEQ ID NO:215), ID1 (SEQ ID NO: 217), GJA1 (SEQ ID NO: 218), IGLC1 (SEQ ID NO:301, 302, 303, or 318), LAPTM5 (SEQ ID NO: 304 or 338), ARHGDIB (SEQ IDNO: 305 or 311), SLC43A3 (SEQ ID NO: 307), LCP1 (SEQ ID NO: 308), HCLS1(SEQ ID NO: 309), CD53 (SEQ ID NO: 310), MZB1 (SEQ ID NO: 313), RASSF5(SEQ ID NO: 314 or 386), FAM46C (SEQ ID NO: 315), RCSD1 (SEQ ID NO:316), IGJ (SEQ ID NO: 317), LPXN (SEQ ID NO: 319), ITGB7 (SEQ ID NO:320), and/or GTSF1 (SEQ ID NO: 321). The expression level of thebiomarker(s) may be determined using, e.g., a microarray, PCR, or othertechniques described herein, for example, using a nucleic acid probesequence based on the target sequences shown in Tables 1-4.

The biomarker PLS3 (SEQ ID NO: 205) may be used to assess a cancerpatient's (e.g, a patient having cancer that is resistant to one or morecancer therapies other than irofulven) responsiveness to irofulven. Theexpression level of the biomarker PLS3 (SEQ ID NO: 205) may be assessedusing nucleic acid amplification methods (e.g., PCR) or a device (e.g.,a microarray). As is described above, the expression level of PLS3 (SEQID NO: 205) in the patient sample may then be compared, e.g., to theexpression level of PLS3 (SEQ ID NO: 205) in a cell (e.g., a cancercell) or tissue (e.g., a tumor tissue) known to be sensitive orresistant to treatment with irofulven and used to determine the cancerpatient's responsiveness to irofulven. The biomarker PLS3 (SEQ ID NO:205) may be used alone to predict cancer patient responsiveness totreatment with irofulven or in combination with one or more additionalbiomarkers (e.g., one, two, three, four, five, ten, or all of thebiomarkers shown in Tables 1-4), such as ATP1B1 (SEQ ID NO: 201 or 219),UCHL1 (SEQ ID NO: 202), PTGR1 (SEQ ID NO: 203 or 210), NME7 (SEQ ID NO:204), S100A10 (SEQ ID NO: 206), CD24 (SEQ ID NO: 207, 209, or 220), NQO1(SEQ ID NO: 208 or 216), MYOF (SEQ ID NO: 211), LAPTM4B (SEQ ID NO:212), CALD1 (SEQ ID NO: 213), PDGFC (SEQ ID NO: 214), BASP1 (SEQ ID NO:215), ID1 (SEQ ID NO: 217), GJA1 (SEQ ID NO: 218), IGLC1 (SEQ ID NO:301, 302, 303, or 318), LAPTM5 (SEQ ID NO: 304 or 338), ARHGDIB (SEQ IDNO: 305 or 311), SLC43A3 (SEQ ID NO: 307), LCP1 (SEQ ID NO: 308), HCLS1(SEQ ID NO: 309), CD53 (SEQ ID NO: 310), MZB1 (SEQ ID NO: 313), RASSF5(SEQ ID NO: 314 or 386), FAM46C (SEQ ID NO: 315), RCSD1 (SEQ ID NO:316), IGJ (SEQ ID NO: 317), LPXN (SEQ ID NO: 319), ITGB7 (SEQ ID NO:320), and/or GTSF1 (SEQ ID NO: 321). The expression level of thebiomarker(s) may be determined using, e.g., a microarray, PCR, or othertechniques described herein, for example, using a nucleic acid probesequence based on the target sequences shown in Tables 1-4.

The biomarker S100A10 (SEQ ID NO: 206) may be used to assess a cancerpatient's (e.g, a patient having cancer that is resistant to one or morecancer therapies other than irofulven) responsiveness to irofulven. Theexpression level of the biomarker S100A10 (SEQ ID NO: 206) may beassessed using nucleic acid amplification methods (e.g., PCR) or adevice (e.g., a microarray). As is described above, the expression levelof S100A10 (SEQ ID NO: 206) in the patient sample may then be compared,e.g., to the expression level of S100A10 (SEQ ID NO: 206) in a cell(e.g., a cancer cell) or tissue (e.g., a tumor tissue) known to besensitive or resistant to treatment with irofulven and used to determinethe cancer patient's responsiveness to irofulven. The biomarker S100A10(SEQ ID NO: 206) may be used alone to predict cancer patientresponsiveness to treatment with irofulven or in combination with one ormore additional biomarkers (e.g., one, two, three, four, five, ten, orall of the biomarkers shown in Tables 1-4), such as ATP1B1 (SEQ ID NO:201 or 219), UCHL1 (SEQ ID NO: 202), PTGR1 (SEQ ID NO: 203 or 210), NME7(SEQ ID NO: 204), PLS3 (SEQ ID NO: 205), CD24 (SEQ ID NO: 207, 209, or220), NQO1 (SEQ ID NO: 208 or 216), MYOF (SEQ ID NO: 211), LAPTM4B (SEQID NO: 212), CALD1 (SEQ ID NO: 213), PDGFC (SEQ ID NO: 214), BASP1 (SEQID NO: 215), ID1 (SEQ ID NO: 217), GJA1 (SEQ ID NO: 218), IGLC1 (SEQ IDNO: 301, 302, 303, or 318), LAPTM5 (SEQ ID NO: 304 or 338), ARHGDIB (SEQID NO: 305 or 311), SLC43A3 (SEQ ID NO: 307), LCP1 (SEQ ID NO: 308),HCLS1 (SEQ ID NO: 309), CD53 (SEQ ID NO: 310), MZB1 (SEQ ID NO: 313),RASSF5 (SEQ ID NO: 314 or 386), FAM46C (SEQ ID NO: 315), RCSD1 (SEQ IDNO: 316), IGJ (SEQ ID NO: 317), LPXN (SEQ ID NO: 319), ITGB7 (SEQ ID NO:320), and/or GTSF1 (SEQ ID NO: 321). The expression level of thebiomarker(s) may be determined using, e.g., a microarray, PCR, or othertechniques described herein, for example, using a nucleic acid probesequence based on the target sequences shown in Tables 1-4.

The biomarker CD24 (SEQ ID NO: 207, 209, or 220) may be used to assess acancer patient's (e.g, a patient having cancer that is resistant to oneor more cancer therapies other than irofulven) responsiveness toirofulven. The expression level of the biomarker CD24 (SEQ ID NO: 207,209, or 220) may be assessed using nucleic acid amplification methods(e.g., PCR) or a device (e.g., a microarray). As is described above, theexpression level of CD24 (SEQ ID NO: 207, 209, or 220) in the patientsample may then be compared, e.g., to the expression level of CD24 (SEQID NO: 207, 209, or 220) in a cell (e.g., a cancer cell) or tissue(e.g., a tumor tissue) known to be sensitive or resistant to treatmentwith irofulven and used to determine the cancer patient's responsivenessto irofulven. The biomarker CD24 (SEQ ID NO: 207, 209, or 220) may beused alone to predict cancer patient responsiveness to treatment withirofulven or in combination with one or more additional biomarkers(e.g., one, two, three, four, five, ten, or all of the biomarkers shownin Tables 1-4), such as ATP1B1 (SEQ ID NO: 201 or 219), UCHL1 (SEQ IDNO: 202), PTGR1 (SEQ ID NO: 203 or 210), NME7 (SEQ ID NO: 204), PLS3(SEQ ID NO: 205), S100A10 (SEQ ID NO: 206), NQO1 (SEQ ID NO: 208 or216), MYOF (SEQ ID NO: 211), LAPTM4B (SEQ ID NO: 212), CALD1 (SEQ ID NO:213), PDGFC (SEQ ID NO: 214), BASP1 (SEQ ID NO: 215), ID1 (SEQ ID NO:217), GJA1 (SEQ ID NO: 218), IGLC1 (SEQ ID NO: 301, 302, 303, or 318),LAPTM5 (SEQ ID NO: 304 or 338), ARHGDIB (SEQ ID NO: 305 or 311), SLC43A3(SEQ ID NO: 307), LCP1 (SEQ ID NO: 308), HCLS1 (SEQ ID NO: 309), CD53(SEQ ID NO: 310), MZB1 (SEQ ID NO: 313), RASSF5 (SEQ ID NO: 314 or 386),FAM46C (SEQ ID NO: 315), RCSD1 (SEQ ID NO: 316), IGJ (SEQ ID NO: 317),LPXN (SEQ ID NO: 319), ITGB7 (SEQ ID NO: 320), and/or GTSF1 (SEQ ID NO:321). The expression level of the biomarker(s) may be determined using,e.g., a microarray, PCR, or other techniques described herein, forexample, using a nucleic acid probe sequence based on the targetsequences shown in Tables 1-4.

The biomarker NQO1 (SEQ ID NO: 208 or 216) may be used to assess acancer patient's (e.g, a patient having cancer that is resistant to oneor more cancer therapies other than irofulven) responsiveness toirofulven. The expression level of the biomarker NQO1 (SEQ ID NO: 208 or216) may be assessed using nucleic acid amplification methods (e.g.,PCR) or a device (e.g., a microarray). As is described above, theexpression level of NQO1 (SEQ ID NO: 208 or 216) in the patient samplemay then be compared, e.g., to the expression level of NQO1 (SEQ ID NO:208 or 216) in a cell (e.g., a cancer cell) or tissue (e.g., a tumortissue) known to be sensitive or resistant to treatment with irofulvenand used to determine the cancer patient's responsiveness to irofulven.The biomarker NQO1 (SEQ ID NO: 208 or 216) may be used alone to predictcancer patient responsiveness to treatment with irofulven or incombination with one or more additional biomarkers (e.g., one, two,three, four, five, ten, or all of the biomarkers shown in Tables 1-4),such as ATP1B1 (SEQ ID NO: 201 or 219), UCHL1 (SEQ ID NO: 202), PTGR1(SEQ ID NO: 203 or 210), NME7 (SEQ ID NO: 204), PLS3 (SEQ ID NO: 205),S100A10 (SEQ ID NO: 206), MYOF (SEQ ID NO: 211), LAPTM4B (SEQ ID NO:212), CALD1 (SEQ ID NO: 213), PDGFC (SEQ ID NO: 214), BASP1 (SEQ ID NO:215), ID1 (SEQ ID NO: 217), GJA1 (SEQ ID NO: 218), IGLC1 (SEQ ID NO:301, 302, 303, or 318), LAPTM5 (SEQ ID NO: 304 or 338), ARHGDIB (SEQ IDNO: 305 or 311), SLC43A3 (SEQ ID NO: 307), LCP1 (SEQ ID NO: 308), HCLS1(SEQ ID NO: 309), CD53 (SEQ ID NO: 310), MZB1 (SEQ ID NO: 313), RASSF5(SEQ ID NO: 314 or 386), FAM46C (SEQ ID NO: 315), RCSD1 (SEQ ID NO:316), IGJ (SEQ ID NO: 317), LPXN (SEQ ID NO: 319), ITGB7 (SEQ ID NO:320), and/or GTSF1 (SEQ ID NO: 321). The expression level of thebiomarker(s) may be determined using, e.g., a microarray, PCR, or othertechniques described herein, for example, using a nucleic acid probesequence based on the target sequences shown in Tables 1-4.

The expression levels of one or more biomarkers of Tables 1-4 can beused to determine cancer patient responsiveness to treatment withirofulven. Once determined to be responsive, the patient can be treatedwith irofulven. In particular, the biomarker MYOF (SEQ ID NO: 211) maybe used to assess a cancer patient's (e.g, a patient having cancer thatis resistant to one or more cancer therapies other than irofulven)responsiveness to irofulven. The expression level of the biomarker MYOF(SEQ ID NO: 211) may be assessed using nucleic acid amplificationmethods (e.g., PCR) or a device (e.g., a microarray). As is describedabove, the expression level of MYOF (SEQ ID NO: 211) in the patientsample may then be compared, e.g., to the expression level of MYOF (SEQID NO: 211) in a cell (e.g., a cancer cell) or tissue (e.g., a tumortissue) known to be sensitive or resistant to treatment with irofulvenand used to determine the cancer patient's responsiveness to irofulven.The biomarker MYOF (SEQ ID NO: 211) may be used alone to predict cancerpatient responsiveness to treatment with irofulven or in combinationwith one or more additional biomarkers (e.g., one, two, three, four,five, ten, or all of the biomarkers shown in Tables 1-4), such as ATP1B1(SEQ ID NO: 201 or 219), UCHL1 (SEQ ID NO: 202), PTGR1 (SEQ ID NO: 203or 210), NME7 (SEQ ID NO: 204), PLS3 (SEQ ID NO: 205), S100A10 (SEQ IDNO: 206), CD24 (SEQ ID NO: 207, 209, or 220), NQO1 (SEQ ID NO: 208 or216), LAPTM4B (SEQ ID NO: 212), CALD1 (SEQ ID NO: 213), PDGFC (SEQ IDNO: 214), BASP1 (SEQ ID NO: 215), ID1 (SEQ ID NO: 217), GJA1 (SEQ ID NO:218), IGLC1 (SEQ ID NO: 301, 302, 303, or 318), LAPTM5 (SEQ ID NO: 304or 338), ARHGDIB (SEQ ID NO: 305 or 311), SLC43A3 (SEQ ID NO: 307), LCP1(SEQ ID NO: 308), HCLS1 (SEQ ID NO: 309), CD53 (SEQ ID NO: 310), MZB1(SEQ ID NO: 313), RASSF5 (SEQ ID NO: 314 or 386), FAM46C (SEQ ID NO:315), RCSD1 (SEQ ID NO: 316), IGJ (SEQ ID NO: 317), LPXN (SEQ ID NO:319), ITGB7 (SEQ ID NO: 320), and/or GTSF1 (SEQ ID NO: 321). Theexpression level of the biomarker(s) may be determined using, e.g., amicroarray, PCR, or other techniques described herein, for example,using a nucleic acid probe sequence based on the target sequences shownin Tables 1-4.

The biomarker LAPTM4B (SEQ ID NO: 212) may be used to assess a cancerpatient's (e.g, a patient having cancer that is resistant to one or morecancer therapies other than irofulven) responsiveness to irofulven. Theexpression level of the biomarker LAPTM4B (SEQ ID NO: 212) may beassessed using nucleic acid amplification methods (e.g., PCR) or adevice (e.g., a microarray). As is described above, the expression levelof LAPTM4B (SEQ ID NO: 212) in the patient sample may then be compared,e.g., to the expression level of LAPTM4B (SEQ ID NO: 212) in a cell(e.g., a cancer cell) or tissue (e.g., a tumor tissue) known to besensitive or resistant to treatment with irofulven and used to determinethe cancer patient's responsiveness to irofulven. The biomarker LAPTM4B(SEQ ID NO: 212) may be used alone to predict cancer patientresponsiveness to treatment with irofulven or in combination with one ormore additional biomarkers (e.g., one, two, three, four, five, ten, orall of the biomarkers shown in Tables 1-4), such as ATP1B1 (SEQ ID NO:201 or 219), UCHL1 (SEQ ID NO: 202), PTGR1 (SEQ ID NO: 203 or 210), NME7(SEQ ID NO: 204), PLS3 (SEQ ID NO: 205), S100A10 (SEQ ID NO: 206), CD24(SEQ ID NO: 207, 209, or 220), NQO1 (SEQ ID NO: 208 or 216), MYOF (SEQID NO: 211), CALD1 (SEQ ID NO: 213), PDGFC (SEQ ID NO: 214), BASP1 (SEQID NO: 215), ID1 (SEQ ID NO: 217), GJA1 (SEQ ID NO: 218), IGLC1 (SEQ IDNO: 301, 302, 303, or 318), LAPTM5 (SEQ ID NO: 304 or 338), ARHGDIB (SEQID NO: 305 or 311), SLC43A3 (SEQ ID NO: 307), LCP1 (SEQ ID NO: 308),HCLS1 (SEQ ID NO: 309), CD53 (SEQ ID NO: 310), MZB1 (SEQ ID NO: 313),RASSF5 (SEQ ID NO: 314 or 386), FAM46C (SEQ ID NO: 315), RCSD1 (SEQ IDNO: 316), IGJ (SEQ ID NO: 317), LPXN (SEQ ID NO: 319), ITGB7 (SEQ ID NO:320), and/or GTSF1 (SEQ ID NO: 321). The expression level of thebiomarker(s) may be determined using, e.g., a microarray, PCR, or othertechniques described herein, for example, using a nucleic acid probesequence based on the target sequences shown in Tables 1-4.

The biomarker CALD1 (SEQ ID NO: 213) may be used to assess a cancerpatient's (e.g, a patient having cancer that is resistant to one or morecancer therapies other than irofulven) responsiveness to irofulven. Theexpression level of the biomarker CALD1 (SEQ ID NO: 213) may be assessedusing nucleic acid amplification methods (e.g., PCR) or a device (e.g.,a microarray). As is described above, the expression level of CALD1 (SEQID NO: 213) in the patient sample may then be compared, e.g., to theexpression level of CALD1 (SEQ ID NO: 213) in a cell (e.g., a cancercell) or tissue (e.g., a tumor tissue) known to be sensitive orresistant to treatment with irofulven and used to determine the cancerpatient's responsiveness to irofulven. The biomarker CALD1 (SEQ ID NO:213) may be used alone to predict cancer patient responsiveness totreatment with irofulven or in combination with one or more additionalbiomarkers (e.g., one, two, three, four, five, ten, or all of thebiomarkers shown in Tables 1-4), such as ATP1B1 (SEQ ID NO: 201 or 219),UCHL1 (SEQ ID NO: 202), PTGR1 (SEQ ID NO: 203 or 210), NME7 (SEQ ID NO:204), PLS3 (SEQ ID NO: 205), S100A10 (SEQ ID NO: 206), CD24 (SEQ ID NO:207, 209, or 220), NQO1 (SEQ ID NO: 208 or 216), MYOF (SEQ ID NO: 211),LAPTM4B (SEQ ID NO: 212), PDGFC (SEQ ID NO: 214), BASP1 (SEQ ID NO:215), ID1 (SEQ ID NO: 217), GJA1 (SEQ ID NO: 218), IGLC1 (SEQ ID NO:301, 302, 303, or 318), LAPTM5 (SEQ ID NO: 304 or 338), ARHGDIB (SEQ IDNO: 305 or 311), SLC43A3 (SEQ ID NO: 307), LCP1 (SEQ ID NO: 308), HCLS1(SEQ ID NO: 309), CD53 (SEQ ID NO: 310), MZB1 (SEQ ID NO: 313), RASSF5(SEQ ID NO: 314 or 386), FAM46C (SEQ ID NO: 315), RCSD1 (SEQ ID NO:316), IGJ (SEQ ID NO: 317), LPXN (SEQ ID NO: 319), ITGB7 (SEQ ID NO:320), and/or GTSF1 (SEQ ID NO: 321). The expression level of thebiomarker(s) may be determined using, e.g., a microarray, PCR, or othertechniques described herein, for example, using a nucleic acid probesequence based on the target sequences shown in Tables 1-4.

The biomarker PDGFC (SEQ ID NO: 214) may be used to assess a cancerpatient's (e.g, a patient having cancer that is resistant to one or morecancer therapies other than irofulven) responsiveness to irofulven. Theexpression level of the biomarker PDGFC (SEQ ID NO: 214) may be assessedusing nucleic acid amplification methods (e.g., PCR) or a device (e.g.,a microarray). As is described above, the expression level of PDGFC (SEQID NO: 214) in the patient sample may then be compared, e.g., to theexpression level of PDGFC (SEQ ID NO: 214) in a cell (e.g., a cancercell) or tissue (e.g., a tumor tissue) known to be sensitive orresistant to treatment with irofulven and used to determine the cancerpatient's responsiveness to irofulven. The biomarker PDGFC (SEQ ID NO:214) may be used alone to predict cancer patient responsiveness totreatment with irofulven or in combination with one or more additionalbiomarkers (e.g., one, two, three, four, five, ten, or all of thebiomarkers shown in Tables 1-4), such as ATP1B1 (SEQ ID NO: 201 or 219),UCHL1 (SEQ ID NO: 202), PTGR1 (SEQ ID NO: 203 or 210), NME7 (SEQ ID NO:204), PLS3 (SEQ ID NO: 205), S100A10 (SEQ ID NO: 206), CD24 (SEQ ID NO:207, 209, or 220), NQO1 (SEQ ID NO: 208 or 216), MYOF (SEQ ID NO: 211),LAPTM4B (SEQ ID NO: 212), CALD1 (SEQ ID NO: 213), BASP1 (SEQ ID NO:215), ID1 (SEQ ID NO: 217), GJA1 (SEQ ID NO: 218), IGLC1 (SEQ ID NO:301, 302, 303, or 318), LAPTM5 (SEQ ID NO: 304 or 338), ARHGDIB (SEQ IDNO: 305 or 311), SLC43A3 (SEQ ID NO: 307), LCP1 (SEQ ID NO: 308), HCLS1(SEQ ID NO: 309), CD53 (SEQ ID NO: 310), MZB1 (SEQ ID NO: 313), RASSF5(SEQ ID NO: 314 or 386), FAM46C (SEQ ID NO: 315), RCSD1 (SEQ ID NO:316), IGJ (SEQ ID NO: 317), LPXN (SEQ ID NO: 319), ITGB7 (SEQ ID NO:320), and/or GTSF1 (SEQ ID NO: 321). The expression level of thebiomarker(s) may be determined using, e.g., a microarray, PCR, or othertechniques described herein, for example, using a nucleic acid probesequence based on the target sequences shown in Tables 1-4.

The biomarker BASP1 (SEQ ID NO: 215) may be used to assess a cancerpatient's (e.g, a patient having cancer that is resistant to one or morecancer therapies other than irofulven) responsiveness to irofulven. Theexpression level of the biomarker BASP1 (SEQ ID NO: 215) may be assessedusing nucleic acid amplification methods (e.g., PCR) or a device (e.g.,a microarray). As is described above, the expression level of BASP1 (SEQID NO: 215) in the patient sample may then be compared, e.g., to theexpression level of BASP1 (SEQ ID NO: 215) in a cell (e.g., a cancercell) or tissue (e.g., a tumor tissue) known to be sensitive orresistant to treatment with irofulven and used to determine the cancerpatient's responsiveness to irofulven. The biomarker BASP1 (SEQ ID NO:215) may be used alone to predict cancer patient responsiveness totreatment with irofulven or in combination with one or more additionalbiomarkers (e.g., one, two, three, four, five, ten, or all of thebiomarkers shown in Tables 1-4), such as ATP1B1 (SEQ ID NO: 201 or 219),UCHL1 (SEQ ID NO: 202), PTGR1 (SEQ ID NO: 203 or 210), NME7 (SEQ ID NO:204), PLS3 (SEQ ID NO: 205), S100A10 (SEQ ID NO: 206), CD24 (SEQ ID NO:207, 209, or 220), NQO1 (SEQ ID NO: 208 or 216), MYOF (SEQ ID NO: 211),LAPTM4B (SEQ ID NO: 212), CALD1 (SEQ ID NO: 213), PDGFC (SEQ ID NO:214), ID1 (SEQ ID NO: 217), GJA1 (SEQ ID NO: 218), IGLC1 (SEQ ID NO:301, 302, 303, or 318), LAPTM5 (SEQ ID NO: 304 or 338), ARHGDIB (SEQ IDNO: 305 or 311), SLC43A3 (SEQ ID NO: 307), LCP1 (SEQ ID NO: 308), HCLS1(SEQ ID NO: 309), CD53 (SEQ ID NO: 310), MZB1 (SEQ ID NO: 313), RASSF5(SEQ ID NO: 314 or 386), FAM46C (SEQ ID NO: 315), RCSD1 (SEQ ID NO:316), IGJ (SEQ ID NO: 317), LPXN (SEQ ID NO: 319), ITGB7 (SEQ ID NO:320), and/or GTSF1 (SEQ ID NO: 321). The expression level of thebiomarker(s) may be determined using, e.g., a microarray, PCR, or othertechniques described herein, for example, using a nucleic acid probesequence based on the target sequences shown in Tables 1-4.

The biomarker ID1 (SEQ ID NO: 217) may be used to assess a cancerpatient's (e.g, a patient having cancer that is resistant to one or morecancer therapies other than irofulven) responsiveness to irofulven. Theexpression level of the biomarker ID1 (SEQ ID NO: 217) may be assessedusing nucleic acid amplification methods (e.g., PCR) or a device (e.g.,a microarray). As is described above, the expression level of ID1 (SEQID NO: 217) in the patient sample may then be compared, e.g., to theexpression level of ID1 (SEQ ID NO: 217) in a cell (e.g., a cancer cell)or tissue (e.g., a tumor tissue) known to be sensitive or resistant totreatment with irofulven and used to determine the cancer patient'sresponsiveness to irofulven. The biomarker ID1 (SEQ ID NO: 217) may beused alone to predict cancer patient responsiveness to treatment withirofulven or in combination with one or more additional biomarkers(e.g., one, two, three, four, five, ten, or all of the biomarkers shownin Tables 1-4), such as ATP1B1 (SEQ ID NO: 201 or 219), UCHL1 (SEQ IDNO: 202), PTGR1 (SEQ ID NO: 203 or 210), NME7 (SEQ ID NO: 204), PLS3(SEQ ID NO: 205), S100A10 (SEQ ID NO: 206), CD24 (SEQ ID NO: 207, 209,or 220), NQO1 (SEQ ID NO: 208 or 216), MYOF (SEQ ID NO: 211), LAPTM4B(SEQ ID NO: 212), CALD1 (SEQ ID NO: 213), PDGFC (SEQ ID NO: 214), BASP1(SEQ ID NO: 215), GJA1 (SEQ ID NO: 218), IGLC1 (SEQ ID NO: 301, 302,303, or 318), LAPTM5 (SEQ ID NO: 304 or 338), ARHGDIB (SEQ ID NO: 305 or311), SLC43A3 (SEQ ID NO: 307), LCP1 (SEQ ID NO: 308), HCLS1 (SEQ ID NO:309), CD53 (SEQ ID NO: 310), MZB1 (SEQ ID NO: 313), RASSF5 (SEQ ID NO:314 or 386), FAM46C (SEQ ID NO: 315), RCSD1 (SEQ ID NO: 316), IGJ (SEQID NO: 317), LPXN (SEQ ID NO: 319), ITGB7 (SEQ ID NO: 320), and/or GTSF1(SEQ ID NO: 321). The expression level of the biomarker(s) may bedetermined using, e.g., a microarray, PCR, or other techniques describedherein, for example, using a nucleic acid probe sequence based on thetarget sequences shown in Tables 1-4.

The biomarker GJA1 (SEQ ID NO: 218) may be used to assess a cancerpatient's (e.g, a patient having cancer that is resistant to one or morecancer therapies other than irofulven) responsiveness to irofulven. Theexpression level of the biomarker GJA1 (SEQ ID NO: 218) may be assessedusing nucleic acid amplification methods (e.g., PCR) or a device (e.g.,a microarray). As is described above, the expression level of GJA1 (SEQID NO: 218) in the patient sample may then be compared, e.g., to theexpression level of GJA1 (SEQ ID NO: 218) in a cell (e.g., a cancercell) or tissue (e.g., a tumor tissue) known to be sensitive orresistant to treatment with irofulven and used to determine the cancerpatient's responsiveness to irofulven. The biomarker GJA1 (SEQ ID NO:218) may be used alone to predict cancer patient responsiveness totreatment with irofulven or in combination with one or more additionalbiomarkers (e.g., one, two, three, four, five, ten, or all of thebiomarkers shown in Tables 1-4), such as ATP1B1 (SEQ ID NO: 201 or 219),UCHL1 (SEQ ID NO: 202), PTGR1 (SEQ ID NO: 203 or 210), NME7 (SEQ ID NO:204), PLS3 (SEQ ID NO: 205), S100A10 (SEQ ID NO: 206), CD24 (SEQ ID NO:207, 209, or 220), NQO1 (SEQ ID NO: 208 or 216), MYOF (SEQ ID NO: 211),LAPTM4B (SEQ ID NO: 212), CALD1 (SEQ ID NO: 213), PDGFC (SEQ ID NO:214), BASP1 (SEQ ID NO: 215), ID1 (SEQ ID NO: 217), IGLC1 (SEQ ID NO:301, 302, 303, or 318), LAPTM5 (SEQ ID NO: 304 or 338), ARHGDIB (SEQ IDNO: 305 or 311), SLC43A3 (SEQ ID NO: 307), LCP1 (SEQ ID NO: 308), HCLS1(SEQ ID NO: 309), CD53 (SEQ ID NO: 310), MZB1 (SEQ ID NO: 313), RASSF5(SEQ ID NO: 314 or 386), FAM46C (SEQ ID NO: 315), RCSD1 (SEQ ID NO:316), IGJ (SEQ ID NO: 317), LPXN (SEQ ID NO: 319), ITGB7 (SEQ ID NO:320), and/or GTSF1 (SEQ ID NO: 321). The expression level of thebiomarker(s) may be determined using, e.g., a microarray, PCR, or othertechniques described herein, for example, using a nucleic acid probesequence based on the target sequences shown in Tables 1-4.

The biomarker IGLC1 (SEQ ID NO: 301, 302, 303, or 318) may be used toassess a cancer patient's (e.g, a patient having cancer that isresistant to one or more cancer therapies other than irofulven)responsiveness to irofulven. The expression level of the biomarker IGLC1(SEQ ID NO: 301, 302, 303, or 318) may be assessed using nucleic acidamplification methods (e.g., PCR) or a device (e.g., a microarray). Asis described above, the expression level of IGLC1 (SEQ ID NO: 301, 302,303, or 318) in the patient sample may then be compared, e.g., to theexpression level of IGLC1 (SEQ ID NO: 301, 302, 303, or 318) in a cell(e.g., a cancer cell) or tissue (e.g., a tumor tissue) known to besensitive or resistant to treatment with irofulven and used to determinethe cancer patient's responsiveness to irofulven. The biomarker IGLC1(SEQ ID NO: 301, 302, 303, or 318) may be used alone to predict cancerpatient responsiveness to treatment with irofulven or in combinationwith one or more additional biomarkers (e.g., one, two, three, four,five, ten, or all of the biomarkers shown in Tables 1-4), such as ATP1B1(SEQ ID NO: 201 or 219), UCHL1 (SEQ ID NO: 202), PTGR1 (SEQ ID NO: 203or 210), NME7 (SEQ ID NO: 204), PLS3 (SEQ ID NO: 205), S100A10 (SEQ IDNO: 206), CD24 (SEQ ID NO: 207, 209, or 220), NQO1 (SEQ ID NO: 208 or216), MYOF (SEQ ID NO: 211), LAPTM4B (SEQ ID NO: 212), CALD1 (SEQ ID NO:213), PDGFC (SEQ ID NO: 214), BASP1 (SEQ ID NO: 215), ID1 (SEQ ID NO:217), GJA1 (SEQ ID NO: 218), LAPTM5 (SEQ ID NO: 304 or 338), ARHGDIB(SEQ ID NO: 305 or 311), SLC43A3 (SEQ ID NO: 307), LCP1 (SEQ ID NO:308), HCLS1 (SEQ ID NO: 309), CD53 (SEQ ID NO: 310), MZB1 (SEQ ID NO:313), RASSF5 (SEQ ID NO: 314 or 386), FAM46C (SEQ ID NO: 315), RCSD1(SEQ ID NO: 316), IGJ (SEQ ID NO: 317), LPXN (SEQ ID NO: 319), ITGB7(SEQ ID NO: 320), and/or GTSF1 (SEQ ID NO: 321). The expression level ofthe biomarker(s) may be determined using, e.g., a microarray, PCR, orother techniques described herein, for example, using a nucleic acidprobe sequence based on the target sequences shown in Tables 1-4.

The biomarker LAPTM5 (SEQ ID NO: 304 or 338) may be used to assess acancer patient's (e.g, a patient having cancer that is resistant to oneor more cancer therapies other than irofulven) responsiveness toirofulven. The expression level of the biomarker LAPTM5 (SEQ ID NO: 304or 338) may be assessed using nucleic acid amplification methods (e.g.,PCR) or a device (e.g., a microarray). As is described above, theexpression level of LAPTM5 (SEQ ID NO: 304 or 338) in the patient samplemay then be compared, e.g., to the expression level of LAPTM5 (SEQ IDNO: 304 or 338) in a cell (e.g., a cancer cell) or tissue (e.g., a tumortissue) known to be sensitive or resistant to treatment with irofulvenand used to determine the cancer patient's responsiveness to irofulven.The biomarker LAPTM5 (SEQ ID NO: 304 or 338) may be used alone topredict cancer patient responsiveness to treatment with irofulven or incombination with one or more additional biomarkers (e.g., one, two,three, four, five, ten, or all of the biomarkers shown in Tables 1-4),such as ATP1B1 (SEQ ID NO: 201 or 219), UCHL1 (SEQ ID NO: 202), PTGR1(SEQ ID NO: 203 or 210), NME7 (SEQ ID NO: 204), PLS3 (SEQ ID NO: 205),S100A10 (SEQ ID NO: 206), CD24 (SEQ ID NO: 207, 209, or 220), NQO1 (SEQID NO: 208 or 216), MYOF (SEQ ID NO: 211), LAPTM4B (SEQ ID NO: 212),CALD1 (SEQ ID NO: 213), PDGFC (SEQ ID NO: 214), BASP1 (SEQ ID NO: 215),ID1 (SEQ ID NO: 217), GJA1 (SEQ ID NO: 218), IGLC1 (SEQ ID NO: 301, 302,303, or 318), ARHGDIB (SEQ ID NO: 305 or 311), SLC43A3 (SEQ ID NO: 307),LCP1 (SEQ ID NO: 308), HCLS1 (SEQ ID NO: 309), CD53 (SEQ ID NO: 310),MZB1 (SEQ ID NO: 313), RASSF5 (SEQ ID NO: 314 or 386), FAM46C (SEQ IDNO: 315), RCSD1 (SEQ ID NO: 316), IGJ (SEQ ID NO: 317), LPXN (SEQ ID NO:319), ITGB7 (SEQ ID NO: 320), and/or GTSF1 (SEQ ID NO: 321). Theexpression level of the biomarker(s) may be determined using, e.g., amicroarray, PCR, or other techniques described herein, for example,using a nucleic acid probe sequence based on the target sequences shownin Tables 1-4.

The biomarker ARHGDIB (SEQ ID NO: 305 or 311) may be used to assess acancer patient's (e.g, a patient having cancer that is resistant to oneor more cancer therapies other than irofulven) responsiveness toirofulven. The expression level of the biomarker ARHGDIB (SEQ ID NO: 305or 311) may be assessed using nucleic acid amplification methods (e.g.,PCR) or a device (e.g., a microarray). As is described above, theexpression level of ARHGDIB (SEQ ID NO: 305 or 311) in the patientsample may then be compared, e.g., to the expression level of ARHGDIB(SEQ ID NO: 305 or 311) in a cell (e.g., a cancer cell) or tissue (e.g.,a tumor tissue) known to be sensitive or resistant to treatment withirofulven and used to determine the cancer patient's responsiveness toirofulven. The biomarker ARHGDIB (SEQ ID NO: 305 or 311) may be usedalone to predict cancer patient responsiveness to treatment withirofulven or in combination with one or more additional biomarkers(e.g., one, two, three, four, five, ten, or all of the biomarkers shownin Tables 1-4), such as ATP1B1 (SEQ ID NO: 201 or 219), UCHL1 (SEQ IDNO: 202), PTGR1 (SEQ ID NO: 203 or 210), NME7 (SEQ ID NO: 204), PLS3(SEQ ID NO: 205), S100A10 (SEQ ID NO: 206), CD24 (SEQ ID NO: 207, 209,or 220), NQO1 (SEQ ID NO: 208 or 216), MYOF (SEQ ID NO: 211), LAPTM4B(SEQ ID NO: 212), CALD1 (SEQ ID NO: 213), PDGFC (SEQ ID NO: 214), BASP1(SEQ ID NO: 215), ID1 (SEQ ID NO: 217), GJA1 (SEQ ID NO: 218), IGLC1(SEQ ID NO: 301, 302, 303, or 318), LAPTM5 (SEQ ID NO: 304 or 338),SLC43A3 (SEQ ID NO: 307), LCP1 (SEQ ID NO: 308), HCLS1 (SEQ ID NO: 309),CD53 (SEQ ID NO: 310), MZB1 (SEQ ID NO: 313), RASSF5 (SEQ ID NO: 314 or386), FAM46C (SEQ ID NO: 315), RCSD1 (SEQ ID NO: 316), IGJ (SEQ ID NO:317), LPXN (SEQ ID NO: 319), ITGB7 (SEQ ID NO: 320), and/or GTSF1 (SEQID NO: 321). The expression level of the biomarker(s) may be determinedusing, e.g., a microarray, PCR, or other techniques described herein,for example, using a nucleic acid probe sequence based on the targetsequences shown in Tables 1-4.

The biomarker SLC43A3 (SEQ ID NO: 307) may be used to assess a cancerpatient's (e.g, a patient having cancer that is resistant to one or morecancer therapies other than irofulven) responsiveness to irofulven. Theexpression level of the biomarker SLC43A3 (SEQ ID NO: 307) may beassessed using nucleic acid amplification methods (e.g., PCR) or adevice (e.g., a microarray). As is described above, the expression levelof SLC43A3 (SEQ ID NO: 307) in the patient sample may then be compared,e.g., to the expression level of SLC43A3 (SEQ ID NO: 307) in a cell(e.g., a cancer cell) or tissue (e.g., a tumor tissue) known to besensitive or resistant to treatment with irofulven and used to determinethe cancer patient's responsiveness to irofulven. The biomarker SLC43A3(SEQ ID NO: 307) may be used alone to predict cancer patientresponsiveness to treatment with irofulven or in combination with one ormore additional biomarkers (e.g., one, two, three, four, five, ten, orall of the biomarkers shown in Tables 1-4), such as ATP1B1 (SEQ ID NO:201 or 219), UCHL1 (SEQ ID NO: 202), PTGR1 (SEQ ID NO: 203 or 210), NME7(SEQ ID NO: 204), PLS3 (SEQ ID NO: 205), S100A10 (SEQ ID NO: 206), CD24(SEQ ID NO: 207, 209, or 220), NQO1 (SEQ ID NO: 208 or 216), MYOF (SEQID NO: 211), LAPTM4B (SEQ ID NO: 212), CALD1 (SEQ ID NO: 213), PDGFC(SEQ ID NO: 214), BASP1 (SEQ ID NO: 215), ID1 (SEQ ID NO: 217), GJA1(SEQ ID NO: 218), IGLC1 (SEQ ID NO: 301, 302, 303, or 318), LAPTM5 (SEQID NO: 304 or 338), ARHGDIB (SEQ ID NO: 305 or 311), LCP1 (SEQ ID NO:308), HCLS1 (SEQ ID NO: 309), CD53 (SEQ ID NO: 310), MZB1 (SEQ ID NO:313), RASSF5 (SEQ ID NO: 314 or 386), FAM46C (SEQ ID NO: 315), RCSD1(SEQ ID NO: 316), IGJ (SEQ ID NO: 317), LPXN (SEQ ID NO: 319), ITGB7(SEQ ID NO: 320), and/or GTSF1 (SEQ ID NO: 321). The expression level ofthe biomarker(s) may be determined using, e.g., a microarray, PCR, orother techniques described herein, for example, using a nucleic acidprobe sequence based on the target sequences shown in Tables 1-4.

The biomarker LCP1 (SEQ ID NO: 308) may be used to assess a cancerpatient's (e.g, a patient having cancer that is resistant to one or morecancer therapies other than irofulven) responsiveness to irofulven. Theexpression level of the biomarker LCP1 (SEQ ID NO: 308) may be assessedusing nucleic acid amplification methods (e.g., PCR) or a device (e.g.,a microarray). As is described above, the expression level of LCP1 (SEQID NO: 308) in the patient sample may then be compared, e.g., to theexpression level of LCP1 (SEQ ID NO: 308) in a cell (e.g., a cancercell) or tissue (e.g., a tumor tissue) known to be sensitive orresistant to treatment with irofulven and used to determine the cancerpatient's responsiveness to irofulven. The biomarker LCP1 (SEQ ID NO:308) may be used alone to predict cancer patient responsiveness totreatment with irofulven or in combination with one or more additionalbiomarkers (e.g., one, two, three, four, five, ten, or all of thebiomarkers shown in Tables 1-4), such as ATP1B1 (SEQ ID NO: 201 or 219),UCHL1 (SEQ ID NO: 202), PTGR1 (SEQ ID NO: 203 or 210), NME7 (SEQ ID NO:204), PLS3 (SEQ ID NO: 205), S100A10 (SEQ ID NO: 206), CD24 (SEQ ID NO:207, 209, or 220), NQO1 (SEQ ID NO: 208 or 216), MYOF (SEQ ID NO: 211),LAPTM4B (SEQ ID NO: 212), CALD1 (SEQ ID NO: 213), PDGFC (SEQ ID NO:214), BASP1 (SEQ ID NO: 215), ID1 (SEQ ID NO: 217), GJA1 (SEQ ID NO:218), IGLC1 (SEQ ID NO: 301, 302, 303, or 318), LAPTM5 (SEQ ID NO: 304or 338), ARHGDIB (SEQ ID NO: 305 or 311), SLC43A3 (SEQ ID NO: 307),HCLS1 (SEQ ID NO: 309), CD53 (SEQ ID NO: 310), MZB1 (SEQ ID NO: 313),RASSF5 (SEQ ID NO: 314 or 386), FAM46C (SEQ ID NO: 315), RCSD1 (SEQ IDNO: 316), IGJ (SEQ ID NO: 317), LPXN (SEQ ID NO: 319), ITGB7 (SEQ ID NO:320), and/or GTSF1 (SEQ ID NO: 321). The expression level of thebiomarker(s) may be determined using, e.g., a microarray, PCR, or othertechniques described herein, for example, using a nucleic acid probesequence based on the target sequences shown in Tables 1-4.

The biomarker HCLS1 (SEQ ID NO: 309) may be used to assess a cancerpatient's (e.g, a patient having cancer that is resistant to one or morecancer therapies other than irofulven) responsiveness to irofulven. Theexpression level of the biomarker HCLS1 (SEQ ID NO: 309) may be assessedusing nucleic acid amplification methods (e.g., PCR) or a device (e.g.,a microarray). As is described above, the expression level of HCLS1 (SEQID NO: 309) in the patient sample may then be compared, e.g., to theexpression level of HCLS1 (SEQ ID NO: 309) in a cell (e.g., a cancercell) or tissue (e.g., a tumor tissue) known to be sensitive orresistant to treatment with irofulven and used to determine the cancerpatient's responsiveness to irofulven. The biomarker HCLS1 (SEQ ID NO:309) may be used alone to predict cancer patient responsiveness totreatment with irofulven or in combination with one or more additionalbiomarkers (e.g., one, two, three, four, five, ten, or all of thebiomarkers shown in Tables 1-4), such as ATP1B1 (SEQ ID NO: 201 or 219),UCHL1 (SEQ ID NO: 202), PTGR1 (SEQ ID NO: 203 or 210), NME7 (SEQ ID NO:204), PLS3 (SEQ ID NO: 205), S100A10 (SEQ ID NO: 206), CD24 (SEQ ID NO:207, 209, or 220), NQO1 (SEQ ID NO: 208 or 216), MYOF (SEQ ID NO: 211),LAPTM4B (SEQ ID NO: 212), CALD1 (SEQ ID NO: 213), PDGFC (SEQ ID NO:214), BASP1 (SEQ ID NO: 215), ID1 (SEQ ID NO: 217), GJA1 (SEQ ID NO:218), IGLC1 (SEQ ID NO: 301, 302, 303, or 318), LAPTM5 (SEQ ID NO: 304or 338), ARHGDIB (SEQ ID NO: 305 or 311), SLC43A3 (SEQ ID NO: 307), LCP1(SEQ ID NO: 308), CD53 (SEQ ID NO: 310), MZB1 (SEQ ID NO: 313), RASSF5(SEQ ID NO: 314 or 386), FAM46C (SEQ ID NO: 315), RCSD1 (SEQ ID NO:316), IGJ (SEQ ID NO: 317), LPXN (SEQ ID NO: 319), ITGB7 (SEQ ID NO:320), and/or GTSF1 (SEQ ID NO: 321). The expression level of thebiomarker(s) may be determined using, e.g., a microarray, PCR, or othertechniques described herein, for example, using a nucleic acid probesequence based on the target sequences shown in Tables 1-4.

The biomarker CD53 (SEQ ID NO: 310) may be used to assess a cancerpatient's (e.g, a patient having cancer that is resistant to one or morecancer therapies other than irofulven) responsiveness to irofulven. Theexpression level of the biomarker CD53 (SEQ ID NO: 310) may be assessedusing nucleic acid amplification methods (e.g., PCR) or a device (e.g.,a microarray). As is described above, the expression level of CD53 (SEQID NO: 310) in the patient sample may then be compared, e.g., to theexpression level of CD53 (SEQ ID NO: 310) in a cell (e.g., a cancercell) or tissue (e.g., a tumor tissue) known to be sensitive orresistant to treatment with irofulven and used to determine the cancerpatient's responsiveness to irofulven. The biomarker CD53 (SEQ ID NO:310) may be used alone to predict cancer patient responsiveness totreatment with irofulven or in combination with one or more additionalbiomarkers (e.g., one, two, three, four, five, ten, or all of thebiomarkers shown in Tables 1-4), such as ATP1B1 (SEQ ID NO: 201 or 219),UCHL1 (SEQ ID NO: 202), PTGR1 (SEQ ID NO: 203 or 210), NME7 (SEQ ID NO:204), PLS3 (SEQ ID NO: 205), S100A10 (SEQ ID NO: 206), CD24 (SEQ ID NO:207, 209, or 220), NQO1 (SEQ ID NO: 208 or 216), MYOF (SEQ ID NO: 211),LAPTM4B (SEQ ID NO: 212), CALD1 (SEQ ID NO: 213), PDGFC (SEQ ID NO:214), BASP1 (SEQ ID NO: 215), ID1 (SEQ ID NO: 217), GJA1 (SEQ ID NO:218), IGLC1 (SEQ ID NO: 301, 302, 303, or 318), LAPTM5 (SEQ ID NO: 304or 338), ARHGDIB (SEQ ID NO: 305 or 311), SLC43A3 (SEQ ID NO: 307), LCP1(SEQ ID NO: 308), HCLS1 (SEQ ID NO: 309), MZB1 (SEQ ID NO: 313), RASSF5(SEQ ID NO: 314 or 386), FAM46C (SEQ ID NO: 315), RCSD1 (SEQ ID NO:316), IGJ (SEQ ID NO: 317), LPXN (SEQ ID NO: 319), ITGB7 (SEQ ID NO:320), and/or GTSF1 (SEQ ID NO: 321). The expression level of thebiomarker(s) may be determined using, e.g., a microarray, PCR, or othertechniques described herein, for example, using a nucleic acid probesequence based on the target sequences shown in Tables 1-4.

The biomarker MZB1 (SEQ ID NO: 313) may be used to assess a cancerpatient's (e.g, a patient having cancer that is resistant to one or morecancer therapies other than irofulven) responsiveness to irofulven. Theexpression level of the biomarker MZB1 (SEQ ID NO: 313) may be assessedusing nucleic acid amplification methods (e.g., PCR) or a device (e.g.,a microarray). As is described above, the expression level of MZB1 (SEQID NO: 313) in the patient sample may then be compared, e.g., to theexpression level of MZB1 (SEQ ID NO: 313) in a cell (e.g., a cancercell) or tissue (e.g., a tumor tissue) known to be sensitive orresistant to treatment with irofulven and used to determine the cancerpatient's responsiveness to irofulven. The biomarker MZB1 (SEQ ID NO:313) may be used alone to predict cancer patient responsiveness totreatment with irofulven or in combination with one or more additionalbiomarkers (e.g., one, two, three, four, five, ten, or all of thebiomarkers shown in Tables 1-4), such as ATP1B1 (SEQ ID NO: 201 or 219),UCHL1 (SEQ ID NO: 202), PTGR1 (SEQ ID NO: 203 or 210), NME7 (SEQ ID NO:204), PLS3 (SEQ ID NO: 205), S100A10 (SEQ ID NO: 206), CD24 (SEQ ID NO:207, 209, or 220), NQO1 (SEQ ID NO: 208 or 216), MYOF (SEQ ID NO: 211),LAPTM4B (SEQ ID NO: 212), CALD1 (SEQ ID NO: 213), PDGFC (SEQ ID NO:214), BASP1 (SEQ ID NO: 215), ID1 (SEQ ID NO: 217), GJA1 (SEQ ID NO:218), IGLC1 (SEQ ID NO: 301, 302, 303, or 318), LAPTM5 (SEQ ID NO: 304or 338), ARHGDIB (SEQ ID NO: 305 or 311), SLC43A3 (SEQ ID NO: 307), LCP1(SEQ ID NO: 308), HCLS1 (SEQ ID NO: 309), CD53 (SEQ ID NO: 310), RASSF5(SEQ ID NO: 314 or 386), FAM46C (SEQ ID NO: 315), RCSD1 (SEQ ID NO:316), IGJ (SEQ ID NO: 317), LPXN (SEQ ID NO: 319), ITGB7 (SEQ ID NO:320), and/or GTSF1 (SEQ ID NO: 321). The expression level of thebiomarker(s) may be determined using, e.g., a microarray, PCR, or othertechniques described herein, for example, using a nucleic acid probesequence based on the target sequences shown in Tables 1-4.

The biomarker RASSF5 (SEQ ID NO: 314 or 386) may be used to assess acancer patient's (e.g, a patient having cancer that is resistant to oneor more cancer therapies other than irofulven) responsiveness toirofulven. The expression level of the biomarker RASSF5 (SEQ ID NO: 314or 386) may be assessed using nucleic acid amplification methods (e.g.,PCR) or a device (e.g., a microarray). As is described above, theexpression level of RASSF5 (SEQ ID NO: 314 or 386) in the patient samplemay then be compared, e.g., to the expression level of RASSF5 (SEQ IDNO: 314 or 386) in a cell (e.g., a cancer cell) or tissue (e.g., a tumortissue) known to be sensitive or resistant to treatment with irofulvenand used to determine the cancer patient's responsiveness to irofulven.The biomarker RASSF5 (SEQ ID NO: 314 or 386) may be used alone topredict cancer patient responsiveness to treatment with irofulven or incombination with one or more additional biomarkers (e.g., one, two,three, four, five, ten, or all of the biomarkers shown in Tables 1-4),such as ATP1B1 (SEQ ID NO: 201 or 219), UCHL1 (SEQ ID NO: 202), PTGR1(SEQ ID NO: 203 or 210), NME7 (SEQ ID NO: 204), PLS3 (SEQ ID NO: 205),S100A10 (SEQ ID NO: 206), CD24 (SEQ ID NO: 207, 209, or 220), NQO1 (SEQID NO: 208 or 216), MYOF (SEQ ID NO: 211), LAPTM4B (SEQ ID NO: 212),CALD1 (SEQ ID NO: 213), PDGFC (SEQ ID NO: 214), BASP1 (SEQ ID NO: 215),ID1 (SEQ ID NO: 217), GJA1 (SEQ ID NO: 218), IGLC1 (SEQ ID NO: 301, 302,303, or 318), LAPTM5 (SEQ ID NO: 304 or 338), ARHGDIB (SEQ ID NO: 305 or311), SLC43A3 (SEQ ID NO: 307), LCP1 (SEQ ID NO: 308), HCLS1 (SEQ ID NO:309), CD53 (SEQ ID NO: 310), MZB1 (SEQ ID NO: 313), FAM46C (SEQ ID NO:315), RCSD1 (SEQ ID NO: 316), IGJ (SEQ ID NO: 317), LPXN (SEQ ID NO:319), ITGB7 (SEQ ID NO: 320), and/or GTSF1 (SEQ ID NO: 321). Theexpression level of the biomarker(s) may be determined using, e.g., amicroarray, PCR, or other techniques described herein, for example,using a nucleic acid probe sequence based on the target sequences shownin Tables 1-4.

The biomarker FAM46C (SEQ ID NO: 315) may be used to assess a cancerpatient's (e.g, a patient having cancer that is resistant to one or morecancer therapies other than irofulven) responsiveness to irofulven. Theexpression level of the biomarker FAM46C (SEQ ID NO: 315) may beassessed using nucleic acid amplification methods (e.g., PCR) or adevice (e.g., a microarray). As is described above, the expression levelof FAM46C (SEQ ID NO: 315) in the patient sample may then be compared,e.g., to the expression level of FAM46C (SEQ ID NO: 315) in a cell(e.g., a cancer cell) or tissue (e.g., a tumor tissue) known to besensitive or resistant to treatment with irofulven and used to determinethe cancer patient's responsiveness to irofulven. The biomarker FAM46C(SEQ ID NO: 315) may be used alone to predict cancer patientresponsiveness to treatment with irofulven or in combination with one ormore additional biomarkers (e.g., one, two, three, four, five, ten, orall of the biomarkers shown in Tables 1-4), such as ATP1B1 (SEQ ID NO:201 or 219), UCHL1 (SEQ ID NO: 202), PTGR1 (SEQ ID NO: 203 or 210), NME7(SEQ ID NO: 204), PLS3 (SEQ ID NO: 205), S100A10 (SEQ ID NO: 206), CD24(SEQ ID NO: 207, 209, or 220), NQO1 (SEQ ID NO: 208 or 216), MYOF (SEQID NO: 211), LAPTM4B (SEQ ID NO: 212), CALD1 (SEQ ID NO: 213), PDGFC(SEQ ID NO: 214), BASP1 (SEQ ID NO: 215), ID1 (SEQ ID NO: 217), GJA1(SEQ ID NO: 218), IGLC1 (SEQ ID NO: 301, 302, 303, or 318), LAPTM5 (SEQID NO: 304 or 338), ARHGDIB (SEQ ID NO: 305 or 311), SLC43A3 (SEQ ID NO:307), LCP1 (SEQ ID NO: 308), HCLS1 (SEQ ID NO: 309), CD53 (SEQ ID NO:310), MZB1 (SEQ ID NO: 313), RASSF5 (SEQ ID NO: 314 or 386), FRCSD1 (SEQID NO: 316), IGJ (SEQ ID NO: 317), LPXN (SEQ ID NO: 319), ITGB7 (SEQ IDNO: 320), and/or GTSF1 (SEQ ID NO: 321). The expression level of thebiomarker(s) may be determined using, e.g., a microarray, PCR, or othertechniques described herein, for example, using a nucleic acid probesequence based on the target sequences shown in Tables 1-4.

The biomarker RCSD1 (SEQ ID NO: 316) may be used to assess a cancerpatient's (e.g, a patient having cancer that is resistant to one or morecancer therapies other than irofulven) responsiveness to irofulven. Theexpression level of the biomarker RCSD1 (SEQ ID NO: 316) may be assessedusing nucleic acid amplification methods (e.g., PCR) or a device (e.g.,a microarray). As is described above, the expression level of RCSD1 (SEQID NO: 316) in the patient sample may then be compared, e.g., to theexpression level of RCSD1 (SEQ ID NO: 316) in a cell (e.g., a cancercell) or tissue (e.g., a tumor tissue) known to be sensitive orresistant to treatment with irofulven and used to determine the cancerpatient's responsiveness to irofulven. The biomarker RCSD1 (SEQ ID NO:316) may be used alone to predict cancer patient responsiveness totreatment with irofulven or in combination with one or more additionalbiomarkers (e.g., one, two, three, four, five, ten, or all of thebiomarkers shown in Tables 1-4), such as ATP1B1 (SEQ ID NO: 201 or 219),UCHL1 (SEQ ID NO: 202), PTGR1 (SEQ ID NO: 203 or 210), NME7 (SEQ ID NO:204), PLS3 (SEQ ID NO: 205), S100A10 (SEQ ID NO: 206), CD24 (SEQ ID NO:207, 209, or 220), NQO1 (SEQ ID NO: 208 or 216), MYOF (SEQ ID NO: 211),LAPTM4B (SEQ ID NO: 212), CALD1 (SEQ ID NO: 213), PDGFC (SEQ ID NO:214), BASP1 (SEQ ID NO: 215), ID1 (SEQ ID NO: 217), GJA1 (SEQ ID NO:218), IGLC1 (SEQ ID NO: 301, 302, 303, or 318), LAPTM5 (SEQ ID NO: 304or 338), ARHGDIB (SEQ ID NO: 305 or 311), SLC43A3 (SEQ ID NO: 307), LCP1(SEQ ID NO: 308), HCLS1 (SEQ ID NO: 309), CD53 (SEQ ID NO: 310), MZB1(SEQ ID NO: 313), RASSF5 (SEQ ID NO: 314 or 386), FAM46C (SEQ ID NO:315), IGJ (SEQ ID NO: 317), LPXN (SEQ ID NO: 319), ITGB7 (SEQ ID NO:320), and/or GTSF1 (SEQ ID NO: 321). The expression level of thebiomarker(s) may be determined using, e.g., a microarray, PCR, or othertechniques described herein, for example, using a nucleic acid probesequence based on the target sequences shown in Tables 1-4.

The biomarker IGJ (SEQ ID NO: 317) may be used to assess a cancerpatient's (e.g, a patient having cancer that is resistant to one or morecancer therapies other than irofulven) responsiveness to irofulven. Theexpression level of the biomarker IGJ (SEQ ID NO: 317) may be assessedusing nucleic acid amplification methods (e.g., PCR) or a device (e.g.,a microarray). As is described above, the expression level of IGJ (SEQID NO: 317) in the patient sample may then be compared, e.g., to theexpression level of IGJ (SEQ ID NO: 317) in a cell (e.g., a cancer cell)or tissue (e.g., a tumor tissue) known to be sensitive or resistant totreatment with irofulven and used to determine the cancer patient'sresponsiveness to irofulven. The biomarker IGJ (SEQ ID NO: 317) may beused alone to predict cancer patient responsiveness to treatment withirofulven or in combination with one or more additional biomarkers(e.g., one, two, three, four, five, ten, or all of the biomarkers shownin Tables 1-4), such as ATP1B1 (SEQ ID NO: 201 or 219), UCHL1 (SEQ IDNO: 202), PTGR1 (SEQ ID NO: 203 or 210), NME7 (SEQ ID NO: 204), PLS3(SEQ ID NO: 205), S100A10 (SEQ ID NO: 206), CD24 (SEQ ID NO: 207, 209,or 220), NQO1 (SEQ ID NO: 208 or 216), MYOF (SEQ ID NO: 211), LAPTM4B(SEQ ID NO: 212), CALD1 (SEQ ID NO: 213), PDGFC (SEQ ID NO: 214), BASP1(SEQ ID NO: 215), ID1 (SEQ ID NO: 217), GJA1 (SEQ ID NO: 218), IGLC1(SEQ ID NO: 301, 302, 303, or 318), LAPTM5 (SEQ ID NO: 304 or 338),ARHGDIB (SEQ ID NO: 305 or 311), SLC43A3 (SEQ ID NO: 307), LCP1 (SEQ IDNO: 308), HCLS1 (SEQ ID NO: 309), CD53 (SEQ ID NO: 310), MZB1 (SEQ IDNO: 313), RASSF5 (SEQ ID NO: 314 or 386), FAM46C (SEQ ID NO: 315), RCSD1(SEQ ID NO: 316), LPXN (SEQ ID NO: 319), ITGB7 (SEQ ID NO: 320), and/orGTSF1 (SEQ ID NO: 321). The expression level of the biomarker(s) may bedetermined using, e.g., a microarray, PCR, or other techniques describedherein, for example, using a nucleic acid probe sequence based on thetarget sequences shown in Tables 1-4.

The biomarker LPXN (SEQ ID NO: 319) may be used to assess a cancerpatient's (e.g, a patient having cancer that is resistant to one or morecancer therapies other than irofulven) responsiveness to irofulven. Theexpression level of the biomarker LPXN (SEQ ID NO: 319) may be assessedusing nucleic acid amplification methods (e.g., PCR) or a device (e.g.,a microarray). As is described above, the expression level of LPXN (SEQID NO: 319) in the patient sample may then be compared, e.g., to theexpression level of LPXN (SEQ ID NO: 319) in a cell (e.g., a cancercell) or tissue (e.g., a tumor tissue) known to be sensitive orresistant to treatment with irofulven and used to determine the cancerpatient's responsiveness to irofulven. The biomarker LPXN (SEQ ID NO:319) may be used alone to predict cancer patient responsiveness totreatment with irofulven or in combination with one or more additionalbiomarkers (e.g., one, two, three, four, five, ten, or all of thebiomarkers shown in Tables 1-4), such as ATP1B1 (SEQ ID NO: 201 or 219),UCHL1 (SEQ ID NO: 202), PTGR1 (SEQ ID NO: 203 or 210), NME7 (SEQ ID NO:204), PLS3 (SEQ ID NO: 205), S100A10 (SEQ ID NO: 206), CD24 (SEQ ID NO:207, 209, or 220), NQO1 (SEQ ID NO: 208 or 216), MYOF (SEQ ID NO: 211),LAPTM4B (SEQ ID NO: 212), CALD1 (SEQ ID NO: 213), PDGFC (SEQ ID NO:214), BASP1 (SEQ ID NO: 215), ID1 (SEQ ID NO: 217), GJA1 (SEQ ID NO:218), IGLC1 (SEQ ID NO: 301, 302, 303, or 318), LAPTM5 (SEQ ID NO: 304or 338), ARHGDIB (SEQ ID NO: 305 or 311), SLC43A3 (SEQ ID NO: 307), LCP1(SEQ ID NO: 308), HCLS1 (SEQ ID NO: 309), CD53 (SEQ ID NO: 310), MZB1(SEQ ID NO: 313), RASSF5 (SEQ ID NO: 314 or 386), FAM46C (SEQ ID NO:315), RCSD1 (SEQ ID NO: 316), IGJ (SEQ ID NO: 317), ITGB7 (SEQ ID NO:320), and/or GTSF1 (SEQ ID NO: 321). The expression level of thebiomarker(s) may be determined using, e.g., a microarray, PCR, or othertechniques described herein, for example, using a nucleic acid probesequence based on the target sequences shown in Tables 1-4.

The biomarker ITGB7 (SEQ ID NO: 320) may be used to assess a cancerpatient's (e.g, a patient having cancer that is resistant to one or morecancer therapies other than irofulven) responsiveness to irofulven. Theexpression level of the biomarker ITGB7 (SEQ ID NO: 320) may be assessedusing nucleic acid amplification methods (e.g., PCR) or a device (e.g.,a microarray). As is described above, the expression level of ITGB7 (SEQID NO: 320) in the patient sample may then be compared, e.g., to theexpression level of ITGB7 (SEQ ID NO: 320) in a cell (e.g., a cancercell) or tissue (e.g., a tumor tissue) known to be sensitive orresistant to treatment with irofulven and used to determine the cancerpatient's responsiveness to irofulven. The biomarker ITGB7 (SEQ ID NO:320) may be used alone to predict cancer patient responsiveness totreatment with irofulven or in combination with one or more additionalbiomarkers (e.g., one, two, three, four, five, ten, or all of thebiomarkers shown in Tables 1-4), such as ATP1B1 (SEQ ID NO: 201 or 219),UCHL1 (SEQ ID NO: 202), PTGR1 (SEQ ID NO: 203 or 210), NME7 (SEQ ID NO:204), PLS3 (SEQ ID NO: 205), S100A10 (SEQ ID NO: 206), CD24 (SEQ ID NO:207, 209, or 220), NQO1 (SEQ ID NO: 208 or 216), MYOF (SEQ ID NO: 211),LAPTM4B (SEQ ID NO: 212), CALD1 (SEQ ID NO: 213), PDGFC (SEQ ID NO:214), BASP1 (SEQ ID NO: 215), ID1 (SEQ ID NO: 217), GJA1 (SEQ ID NO:218), IGLC1 (SEQ ID NO: 301, 302, 303, or 318), LAPTM5 (SEQ ID NO: 304or 338), ARHGDIB (SEQ ID NO: 305 or 311), SLC43A3 (SEQ ID NO: 307), LCP1(SEQ ID NO: 308), HCLS1 (SEQ ID NO: 309), CD53 (SEQ ID NO: 310), MZB1(SEQ ID NO: 313), RASSF5 (SEQ ID NO: 314 or 386), FAM46C (SEQ ID NO:315), RCSD1 (SEQ ID NO: 316), IGJ (SEQ ID NO: 317), LPXN (SEQ ID NO:319), and/or GTSF1 (SEQ ID NO: 321). The expression level of thebiomarker(s) may be determined using, e.g., a microarray, PCR, or othertechniques described herein, for example, using a nucleic acid probesequence based on the target sequences shown in Tables 1-4.

The biomarker GTSF1 (SEQ ID NO: 321) may be used to assess a cancerpatient's (e.g, a patient having cancer that is resistant to one or morecancer therapies other than irofulven) responsiveness to irofulven. Theexpression level of the biomarker GTSF1 (SEQ ID NO: 321) may be assessedusing nucleic acid amplification methods (e.g., PCR) or a device (e.g.,a microarray). As is described above, the expression level of GTSF1 (SEQID NO: 321) in the patient sample may then be compared, e.g., to theexpression level of GTSF1 (SEQ ID NO: 321) in a cell (e.g., a cancercell) or tissue (e.g., a tumor tissue) known to be sensitive orresistant to treatment with irofulven and used to determine the cancerpatient's responsiveness to irofulven. The biomarker GTSF1 (SEQ ID NO:321) may be used alone to predict cancer patient responsiveness totreatment with irofulven or in combination with one or more additionalbiomarkers (e.g., one, two, three, four, five, ten, or all of thebiomarkers shown in Tables 1-4), such as ATP1B1 (SEQ ID NO: 201 or 219),UCHL1 (SEQ ID NO: 202), PTGR1 (SEQ ID NO: 203 or 210), NME7 (SEQ ID NO:204), PLS3 (SEQ ID NO: 205), S100A10 (SEQ ID NO: 206), CD24 (SEQ ID NO:207, 209, or 220), NQO1 (SEQ ID NO: 208 or 216), MYOF (SEQ ID NO: 211),LAPTM4B (SEQ ID NO: 212), CALD1 (SEQ ID NO: 213), PDGFC (SEQ ID NO:214), BASP1 (SEQ ID NO: 215), ID1 (SEQ ID NO: 217), GJA1 (SEQ ID NO:218), IGLC1 (SEQ ID NO: 301, 302, 303, or 318), LAPTM5 (SEQ ID NO: 304or 338), ARHGDIB (SEQ ID NO: 305 or 311), SLC43A3 (SEQ ID NO: 307), LCP1(SEQ ID NO: 308), HCLS1 (SEQ ID NO: 309), CD53 (SEQ ID NO: 310), MZB1(SEQ ID NO: 313), RASSF5 (SEQ ID NO: 314 or 386), FAM46C (SEQ ID NO:315), RCSD1 (SEQ ID NO: 316), IGJ (SEQ ID NO: 317), LPXN (SEQ ID NO:319), and/or ITGB7 (SEQ ID NO: 320). The expression level of thebiomarker(s) may be determined using, e.g., a microarray, PCR, or othertechniques described herein, for example, using a nucleic acid probesequence based on the target sequences shown in Tables 1-4.

Methods of Treatment

The diagnostic methods of the invention permit the assessment of whethera patient is likely to be responsive to treatment with irofulven, andcan thus be used to direct the patient's treatment (e.g., as a firstline therapy and/or as a second or third line therapy). A patient to betreated or tested for responsiveness to irofulven according to themethods may include, e.g., a patient that has been diagnosed withcancer, a patient that has not received a cancer treatment (e.g.,irofulven, an anti-cancer agent other than irofulven, or radiation), apatient that has received a cancer treatment (e.g., an anti-cancer agentother than irofulven or radiation), or a patient during treatment withirofulven.

For example, the patient may have a solid tumor or a hematologicalcancer, such as a cancer type selected from prostate cancer, ovariancancer (e.g., ovarian adenocarcinoma or embryonal carcinoma), livercancer (e.g., hepatocellular carcinoma (HCC) or hepatoma), myeloma(e.g., multiple myeloma), colorectal cancer (e.g., colon cancer andrectal cancer), leukemia (e.g., acute myeloid leukemia, acute lymphoidleukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, acutemyeloblastic leukemia, acute promyelocytic leukemia, acutemyelomonocytic leukemia, acute monocytic leukemia, acuteerythroleukemia, and chronic leukemia), myelodysplastic syndrome,lymphoma (e.g., diffuse large B-cell lymphoma, cutaneous T-celllymphoma, peripheral T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin'slymphoma, Waldenstrom's macroglobulinemia, and lymphocytic lymphoma),cervical cancer, esophageal cancer, melanoma, glioma (e.g.,oligodendroglioma), pancreatic cancer (e.g., adenosquamous carcinoma,signet ring cell carcinoma, hepatoid carcinoma, colloid carcinoma, isletcell carcinoma, and pancreatic neuroendocrine carcinoma),gastrointestinal stromal tumor, sarcoma (e.g., fibrosarcoma,myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma,angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, leiomyosarcoma, Ewing's sarcoma, andrhabdomyosarcoma), breast cancer (e.g., medullary carcinoma),ER-positive cancer, bladder cancer, head and neck cancer (e.g., squamouscell carcinoma of the head and neck), lung cancer (e.g., non-small celllung carcinoma, large cell carcinoma, bronchogenic carcinoma, andpapillary adenocarcinoma), metastatic cancer, oral cavity cancer,uterine cancer, testicular cancer (e.g., seminoma and embryonalcarcinoma), skin cancer (e.g., squamous cell carcinoma and basal cellcarcinoma), thyroid cancer (e.g., papillary carcinoma and medullarycarcinoma), brain cancer (e.g., astrocytoma and craniopharyngioma),stomach cancer, intra-epithelial cancer, bone cancer, biliary tractcancer, eye cancer, larynx cancer, kidney cancer (e.g., renal cellcarcinoma and Wilms tumor), gastric cancer, blastoma (e.g.,nephroblastoma, medulloblastoma, hemangioblastoma, neuroblastoma, andretinoblastoma), polycythemia vera, chordoma, synovioma, mesothelioma,adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma,cystadenocarcinoma, bile duct carcinoma, choriocarcinoma, epithelialcarcinoma, ependymoma, pinealoma, acoustic neuroma, schwannoma,meningioma, pituitary adenoma, nerve sheath tumor, cancer of the smallintestine, cancer of the endocrine system, cancer of the penis, cancerof the urethra, cutaneous or intraocular melanoma, a gynecologic tumor,solid tumors of childhood, and neoplasms of the central nervous system.In particular, the cancer of the patient is, e.g., prostate cancer,ovarian cancer, hepatocellular carcinoma (HCC), multiple myeloma, breastcancer, acute myelogenous leukemia (AML), acute lympho-blastic leukemia(ALL), chronic lymphocytic leukemia (CLL), myelodysplastic syndrome(MDS), chronic myelogenous leukemia-chronic phase (CMLCP), diffuse largeB-cell lymphoma (DLBCL), cutaneous T-cell lymphoma (CTCL), peripheralT-cell lymphoma (PTCL), Hodgkin's lymphoma, cervical cancer, renal cellcarcinoma (RCC), esophageal cancer, melanoma, glioma, pancreatic cancer,gastrointestinal stromal tumors (GIST), sarcoma, estrogenreceptor-positive (ERpos) breast cancer, non-small cell lung carcinoma(NSCLC), colon cancer, bladder cancer, or squamous cell carcinoma of thehead and neck (SCCHN).

The patient may have a cancer (e.g., prostate cancer, ovarian cancer, orhepatocellular carcinoma (HCC)) that is resistant to one or more cancertherapies other than irofulven (e.g., docetaxel, cabazitaxel,mitoxantrone, estramustine, prednisone, carboplatin, bevacizumab,paclitaxel, gemcitabine, doxorubicin, topotecan, etoposide, tamoxifen,letrozole, sorafenib, fluorouracil, capecitabine, oxaliplatin,interferon-alpha, 5-fluorouracil (5-FU), a histone deacetylase (HDAC)inhibitor, ipilimumab, bortezomib, carfilzomib, thalidomide,lenalidomide, pomalidomide, dexamethasone, cyclophosphamide,vincristine, melphalan, tegafur, irinotecan, cetuximab, leucovorin,SN-38, everolimus, temsirolimus, bleomycin, lomustine, depsipeptide,erlotinib, cisplatin, busulfan, epirubicin, arsenic trioxide,bendamustine, fulvestrant, teniposide, adriamycin, decitabine,estramustine, azaguanine, aclarubicin, mitomycin, paclitaxel, taxotere,APO010, ara-c, methylprednisolone, methotrexate, methyl-gag, belinostat,idarubicin, IL4-PR38, valproic acid, all-trans retinoic acid (ATRA),cytoxan, suberoylanilide hydroxamic acid, leukeran, fludarabine,vinblastine, dacarbazine, hydroxyurea, tegafur, daunorubicin,mechlorethamine, streptozocin, carmustine, mercaptopurine, dactinomycin,tretinoin, ifosfamide, floxuridine, thioguanine, PSC 833, herceptin,celecoxib, iressa, anastrozole, and rituximab), surgery, or radiation.The patient may also have experienced a recurrence following a treatmentwith a cancer therapy other than irofulven, surgery, or radiation.

A patient found to be responsive to irofulven according to the methodsof the invention may be preferentially selected for treatment withirofulven. For example, a patient can be identified as responsive toirofulven by determining the expression level of one or more biomarkers(e.g., one or more of the biomarkers shown in Tables 1-4, such as ATP1B1(SEQ ID NO: 201)) in a biological sample (e.g., a tumor sample) obtainedfrom the patient, and subsequently administered irofulven.Alternatively, a patient can be identified as less likely to beresponsive to irofulven by determining the expression level of one ormore biomarkers (e.g., one or more of the biomarkers shown in Tables1-4, such as ATP1B1 (SEQ ID NO: 201)) in a biological sample obtainedfrom the patient. If the patient exhibits expression levels of one ormore biomarkers indicative of non-responsiveness to irofulven, thepatient may be treated with or offered a treatment with an agent otherthan irofulven. In particular, the patient may be treated with, e.g.,radiation and/or administration of a therapeutic agent, such asdocetaxel, cabazitaxel, mitoxantrone, estramustine, prednisone,carboplatin, bevacizumab, paclitaxel, gemcitabine, doxorubicin,topotecan, etoposide, tamoxifen, letrozole, sorafenib, fluorouracil,capecitabine, oxaliplatin, interferon-alpha, 5-fluorouracil (5-FU), ahistone deacetylase (HDAC) inhibitor, ipilimumab, bortezomib,carfilzomib, thalidomide, lenalidomide, pomalidomide, dexamethasone,cyclophosphamide, vincristine, melphalan, tegafur, irinotecan,cetuximab, leucovorin, SN-38, everolimus, temsirolimus, bleomycin,lomustine, depsipeptide, erlotinib, cisplatin, busulfan, epirubicin,arsenic trioxide, bendamustine, fulvestrant, teniposide, adriamycin,decitabine, estramustine, azaguanine, aclarubicin, mitomycin,paclitaxel, taxotere, APO010, ara-c, methylprednisolone, methotrexate,methyl-gag, belinostat, idarubicin, IL4-PR38, valproic acid, all-transretinoic acid (ATRA), cytoxan, suberoylanilide hydroxamic acid,leukeran, fludarabine, vinblastine, dacarbazine, hydroxyurea, tegafur,daunorubicin, mechlorethamine, streptozocin, carmustine, mercaptopurine,dactinomycin, tretinoin, ifosfamide, floxuridine, thioguanine, PSC 833,herceptin, celecoxib, iressa, anastrozole, or rituximab.

Administration of Irofulven

Once a patient has been determined to be responsive to irofulven,according to the methods described herein, irofulven may be administeredto the patient, for example, parenterally, enterally, or topically.Enteral routes of irofulven administration include oral, buccal,sublabial, sublingual, or by inhalation. Parenteral routes of irofulvenadministration include intravenous, transdermal, intradermal,intramuscular, intra-arterial, intracranial, subcutaneous, intraorbital,intraventricular, intraspinal, intraperitoneal, or intranasal. Thepreferred route for administration of irofulven may be intravenous, suchas intravenous infusion.

Irofulven can be administered at, e.g., a dose of about 0.05 mg/kg to 5mg/kg, about 0.06 mg/kg to 4 mg/kg, about 0.1 mg/kg to 3 mg/kg, about0.2 mg/kg to 2 mg/kg, about 0.3 mg/kg to 1 mg/kg, about 0.4 mg/kg to 1mg/kg, about 0.2 mg/kg to 4 mg/kg, about 0.35 mg/kg to 4.5 mg/kg, about0.25 mg/kg to 3.5 mg/kg, about 0.15 mg/kg to 2.5 mg/kg, about 0.45 mg/kgto about 1 mg/kg, about 0.45 mg/kg to about 2 mg/kg, or about 0.45 mg/kgto about 4 mg/kg. Irofulven may be administered at a frequency of, e.g.,at least once hourly, once daily, twice daily, once weekly, once everytwo weeks, once every three weeks, once every four weeks, once monthly,once every two months, once every three months, once every six months,or once every year.

For example, irofulven may be administered at a treatment regimen of,e.g., about 0.05 mg/kg to 5 mg/kg, about 0.06 mg/kg to 4 mg/kg, about0.1 mg/kg to 3 mg/kg, about 0.2 mg/kg to 2 mg/kg, about 0.3 mg/kg to 1mg/kg, about 0.4 mg/kg to 1 mg/kg, about 0.2 mg/kg to 4 mg/kg, about0.35 mg/kg to 4.5 mg/kg, about 0.25 mg/kg to 3.5 mg/kg, about 0.15 mg/kgto 2.5 mg/kg, about 0.45 mg/kg to about 1 mg/kg, about 0.45 mg/kg toabout 2 mg/kg, or about 0.45 mg/kg to about 4 mg/kg on day 1 and day 8every 3 weeks. In particular, irofulven may be administered at atreatment regimen of about 0.45 mg/kg on day 1 and day 8 every 3 weeks.The treatment regimen may be repeated one to five times, one to tentimes, one to fifteen times, one to twenty times, or more. Theadministration of irofulven can be repeated at such a frequency for acertain period of time, followed by a period without treatment. Suchrepeated administrations can occur over a course of therapy lasting aspecified length of time (e.g., at least 1 week, 2 weeks, 3 weeks, 1month, 2 months, 3 months, 6 months, 8 months, 10 months, 12 months, 18months, 24 months, 36 months, 48 months, or 60 months).

Irofulven can be administered in a pharmaceutical composition thatincludes one or more pharmaceutically acceptable carriers, excipients,or diluents. Examples of suitable carriers, excipients, or diluents ofirofulven include, e.g., saline, sterile water, polyalkylene glycols,oils of vegetable origin, hydrogenated napthalenes, suitable buffer,1,3-butanediol, Ringer's solution and/or sodium chloride solution.Exemplary formulations for parenteral administration can includesolutions prepared in water suitably mixed with a surfactant, e.g.,hydroxypropylcellulose. Dispersions can also be prepared in glycerol,liquid polyethylene glycols, DMSO and mixtures thereof with or withoutalcohol, and in oils. Under ordinary conditions of storage and use,these preparations may contain a preservative to prevent the growth ofmicroorganisms. Other exemplary carriers, excipients, or diluents aredescribed in the Handbook of Pharmaceutical Excipients, 6th Edition,Rowe et al., Eds., Pharmaceutical Press (2009), hereby incorporated byreference in its entirety.

Kits

Kits of the invention can be used for determining the responsiveness ofa cancer patient (e.g., a patient having a solid tumor cancer, such asprostate cancer, ovarian cancer, or hepatocellular carcinoma (HCC), or ahematological cancer, such as lymphoma (e.g., cutaneous T-cell lymphoma(CTCL)) to irofulven. Kits of the invention can include reagents and/ormaterials for, e.g., collecting and/or purifying nucleic acids frombiological samples (such as those obtained from a patient to be treatedwith a target drug(s) of the invention), reagents for amplifying suchnucleic acids to produce an amplified sample, and/or at least one deviceof the invention. Reagents for amplifying nucleic acids may include,e.g., PCR reagents, including but not limited to DNA polymerase, RNApolymerase, PCR buffer, magnesium chloride solutions, nucleic acidprimers (e.g., primers designed to target particular biomarkers ofresponsiveness to a target drug(s) of interest), and/or any other PCRreagents as are well known in the art. In particular, kits useful in themethod may include includes one or more of the following: a kit for RNAextraction from tumors (e.g., Trizol for mRNA, mirVana miRNA isolationkit from Ambion Inc), a kit for RNA labeling (e.g., MessageAmp fromAmbion Inc., FlashTag from Genisphere Inc), a microarray for measuringbiomarker expression (e.g., HG-U133A, HG-U133_Plus2 or miRNA-1.0 fromAffymetrix Inc), a microarray hybridization station and scanner (e.g.,GeneChip System 3000Dx from Affymetrix Inc), and/or software foranalyzing the expression of biomarker genes or RNAs (e.g., miRNAs) asdescribed in herein (e.g., implemented in R from R-Project or S-Plusfrom Insightful Corp.).

For example, a kit of the invention can include one or more probescapable of detecting one or more biomarkers of Tables 1-4 (e.g., the kitmay include probes for the biomarkers of Tables 1-4). Such probes can,for example, include nucleic acids capable of hybridizing to thebiomarker based on nucleic acid sequence complementarity. In particular,a probe has at least 85% sequence identity (e.g., 85%, 90%, 95%, 97%,98%, 99%, or 100% sequence identity) to a nucleic acid sequence that iscomplementary or identical to at least 5 (e.g., at least 15) consecutivenucleotides of one or more biomarkers. The probes can be attached asolid surface, such as a microarray. The kit may include NanoStringcapture probes, NanoString reporter probes, and/or one or more nCountercartridges. The kit may include reagents for next generation sequencing,including but not limited to poly(T) oligonucleotides, dye terminators,sequencing adapters, adapter ligation reagents, reverse transcriptase,primers (e.g., random primers), DNA-cleaving enzymes, polymerases,and/or any combination thereof. The kit may also be one that includes aprotein array and/or reagents for detection of the polypeptideproduct(s) of one or more biomarkers of Tables 1-4.

The following examples are intended to illustrate, rather than limit,the invention.

EXAMPLES Example 1. Identification of Biomarkers of Sensitivity andResistance to Irofulven Using Affymetrix HG-U133A Arrays

DNA chip measurements of the 60 cancer cell lines of the NCI60 data setwere performed using Affymetrix HG-U133A arrays and logit normalized.For each array, the logit transformation was performed followed by aZ-transformation to mean zero and SD 1, and correlated to growthinhibition (log(GI50)). Growth inhibition data of irofulven against thesame cell lines were downloaded from the National Cancer Institute. Eachgene's expression in each cell line was correlated to the growth ofthose cell lines (log(GI50)) in the presence of irofulven. The Pearsoncorrelation coefficient was then determined to identify genes positivelyand negatively correlated to sensitivity to irofulven. Tables 1 and 2show the top positively correlated genes (the biomarkers of sensitivity)and negatively correlated genes (the biomarkers of resistance) using theAffymetrix HG-U133A arrays.

TABLE 1 Biomarkers of sensitivity to irofulven. Dashes indicate that theAffymetrix probeset has not been mapped to a specific gene. AffymetrixIDs refer to the array type HG-U133A. Affymetrix Probe Gene AffymetrixID Correlation Affymetrix Probe Sequence SEQ ID NO: S100A10 200872_at0.571 AGAGTTGTCCAAAGGGTCGCTTAAG 1 ATP1B1 201242_s_at 0.554TGATCACAAGCACAAATCTTTCCCA 2 CTBP2 201220_x_at 0.549ACACCCTGCTCTACAATGTTGCATC 3 ASPH 209135_at 0.539AAAGCTGTGCTGTCGGTGATACAGA 4 LAPTM4B 214039_s_at 0.525GTTACAAAGTCAGCAACTCTCCTGT 5 CTBP2 210835_s_at 0.524TGCACGCATAGGATTGAAGACAGTA 6 MYOF 201798_s_at 0.519CCAGTAGCAGGTTACGCCATGTCAC 7 TJP1 202011_at 0.518AATACACCACTACATTCTGTTAATC 8 ASPH 210896_s_at 0.517GTAACGCCTTGAGCTGGGTTGATTG 9 LPP 202822_at 0.511GTTTGGTGCACTCTCGTGGGAGACA 10 PFN2 204992_s_at 0.503CCCCAGGGACATTCCATCATTGCAA 11 ACTN1 208636_at 0.499CAAGCACAAAGTTATATTCCATCCT 12 CKAP4 200999_s_at 0.474TGTGTAATTGTTTGCTGTATCTCCC 13 MYOF 211864_s_at 0.46TACCAAATCGACCAGAAACCTCCTT 14 PPIC 204517_at 0.455CACCTGCTTAGGGACTTTGAACTTA 15 MET 203510_at 0.454AAACTTGTCCTTAGATTAATGTGTC 16 CEBPD 203973_s_at 0.454AAGCGGCGCAACCAGGAGATGCAGC 17 PAPSS2 203060_s_at 0.452GATTTCAAGCTGTTCTGAGACATCT 18 UGDH 203343_at 0.448TTAATTTCCAGTCACCCCAAATATG 19 FKBP9 212169_at 0.448AACAAGGTGACATTTTTCTGCTGCC 20 NQO1 210519_s_at 0.446GTGGCTTCCAAGTCTTAGAACCTCA 21 EGFR 201983_s_at 0.443AAAATCCAGACTCTTTCGATACCCA 22 LAPTM4B 208767_s_at 0.44AACTTCCCCCAAATCTGATGGACCT 23 RBPMS 209488_s_at 0.44AATTCCGCAAACACTACGACTAGAG 24 ANXA2 210427_x_at 0.439CGTGGCCATCCCTGTGAGGGTGACG 25 NQO1 201468_s_at 0.435TTCATCTTCACTGCAATTTTGTGTA 26 LAPTM4B 208029_s_at 0.433ACATGGGGTGACATGCCTCGTATGT 27 ANXA2 213503_x_at 0.433CTCACCATGCTTCCAGCTAACAGGT 28 ANXA2 201590_x_at 0.43GAGGGTGACGTTAGCATTACCCCCA 29 SEPT10 212698_s_at 0.43TGATGACTTCATGCTTTATTATGCC 30 ALCAM 201952_at 0.425ACAGCTGTCAGAACCTCGAGAGCAG 31 NQO1 201467_s_at 0.423TCTCTGCAAGGGATCCACGGGGACA 32 SKAP2 204362_at 0.422GCAAATGGCAGTGTGCTGTCACCTG 33 CAPN2 208683_at 0.422GACACGAGGCCCTTGGCAGGGAATA 34 RCN1 201063_at 0.42ACAATGCATTTCCTCAGTGATCACT 35 TXNRD1 201266_at 0.418ACACGTGCTTGTGGACATCAGCCTC 36 ACTN1 208637_x_at 0.418GGCGCTGTACGGCGAGAGTGACCTC 37 CALM1 209563_x_at 0.416TGGCCAGTTTTTCATGCATGCAGCT 38 CALM2 CALM3 LAMB1 201505_at 0.412AAGAGTCAGCTGATGCCAGAAGGAA 39 CTSL1 202087_s_at 0.411GAAACCATTGTGGAATTGCCTCAGC 40 TGIF1 203313_s_at 0.41ACTTACAGCTTAACCCATTTTCAAG 41 ANXA2P2 208816_x_at 0.41AAGCCCTTGTATTTTGCTGATCAGC 42 CALM1 200653_s_at 0.408GAAGAAATCCGTGAGGCATTCCGAG 43 CALM2 CALM3 CD24 209771_x_at 0.403GAACACTCTTGCTTTATTCCAGAAT 44 CALM1 211984_at 0.403AATTTGGTCAAGTCTACTCTTCCGT 45 CALM2 CALM3 PLXNB2 208890_s_at 0.397AGAAAAAGCGGTACGATGCCTTCCT 46 SDC4 202071_at 0.396CAGTAACCACATGCGGCTGTTTAAA 47 ACTN4 200601_at 0.394TAACCAAGGAGGGGCCAGTGGATTC 48 TNFRSF12A 218368_s_at 0.389AACACTAGGGGCTGGCCCACTAGGA 49 LOC100652 201506_at 0.386CTTTTATGGGGCCCTGTCCAGGTAG 50 886 LOC100653 157TGFBI SQSTM1 201471_s_at0.385 CTGCCTTCTTCCAGGATCAGGGGTT 51 CD24 216379_x_at 0.384AATAGACACTCCCCGAAGTCTTTTG 52 ALDH7A1 208951_at 0.381ACACAGAGACTTTTGCTCCGATTCT 53 LIF 205266_at 0.379GTAGCATTTCCCTGCAGATGGTACA 54 AMOTL2 203002_at 0.377TTTTTAGTTCTCCTTGATTCTTTGT 55 MYO1B 212364_at 0.377ATGACCAGGTTAAATCCCTCTACAT 56 CDC42BPA 214464_at 0.377AGCTCTGCAGTATTCAACTGTTGAT 57 CCND1 208712_at 0.374AACCAAAAGAATTTGCACCCCGCTG 58 AGRN 212285_s_at 0.374GAGCTCACTGTGGGATGGGGTTGAC 59 CD24 266_s_at 0.373GTAATCTAATATGGCCACAGTAGTC 60 CD24 208651_x_at 0.372AATAGCCACATTTAGAACACTTTTT 61 CD24 208650_s_at 0.371AATCTGCTGGAGTTTCATGTACAAG 62 RBPMS 209487_at 0.37AATTAGATTTGTCTCTGGGAATGTG 63 CALM1 200655_s_at 0.368AACCATCAACATTGCTGTTCAAAGA 64 CALM2 CALM3 LGALS3 208949_s_at 0.367CACTTTAACCCACGCTTCAATGAGA 65 CALM1 211985_s_at 0.367TCGGCAACTTACACACATTGAAAAT 66 CALM2 CALM3 PPP1R3C 204284_at 0.365TGTACACAACTCACATCCTTCATAT 67 LMNA 212086_x_at 0.362TTCGGGGACAATCTGGTCACCCGCT 68 ADAM9 202381_at 0.361ATCTGAACTTTCAAAGCTTGCTATT 69 FZD6 203987_at 0.361GAGTGTCCACTATTGATTGTATTAT 70 TMSB10 217733_s_at 0.361GCAGGAGAAGAACACCCTGCCGACC 71 AMIGO2 222108_at 0.359ATATTGGTTCATGTGCTTGTGTATA 72 PLK2 201939_at 0.358AGCTTTTGGCTGCGTAACTGTGAAC 73 NFIB 209289_at 0.357TGAACGAATTTATTTTCCCCTCAGT 74 ULK2 204062_s_at 0.355ACAGTTCACCAAATAGCTAGTCATG 75 AVPI1 218631_at 0.354CATTTCACCACTCATGCTACTAATC 76 CYR61 201289_at 0.353GTGGAGTTGATGACTTTCTGTTTTC 77 EXT1 201995_at 0.353GGCCAATGAGAACTCAACTCCTGGC 78 TM4SF1 209386_at 0.353AAAGCCTTTTGTCCTCCAAAGATGA 79 COTL1 221059_s_at 0.351TGCAAGCTGCGAGGATGGCTTGGGC 80 PRSS23 202458_at 0.35GAGGGGTGACACAGTGTTCCCTCCT 81 YES1 202933_s_at 0.348AGCTTTTCGTCTTCAGTGTCTTAAT 82 G6PD 202275_at 0.347CCAGAGCTTATTGGCCACTGGGTCT 83 BASP1 202391_at 0.347CATTCTTCCTCTCCAGATATTTTTG 84 ZFP36L1 211962_s_at 0.347ATGGTTTTTGCTCTAGAATACCGTA 85 FAIM 220643_s_at 0.345GGACCAAAGTCACTAATGTTTTACA 86 FLNB 208614_s_at 0.344TCAGCCTGGGCAGTCTTACCAAAAT 87 CAV2 203323_at 0.343CCTAGGCCATTGCAGCATCCTTAGA 88 ABCC3 208161_s_at 0.343CACCCCTAGGAACTCAGTCCTGTAC 89 MIR21 220990_s_at 0.342TAAGGTAAGGTATCCACCCTCGATG 90 VMP1 FTH1 214211_at 0.341TAGAACTAAGGGTTCCCGACTCTGA 91 PLOD2 202620_s_at 0.34TCCATTTTATTCTTCTGAGTGTCTA 92 MXRA7 212509_s_at 0.34CAGCATCAACCCCTATGGCATGCAT 93 ANXA4 201301_s_at 0.339AAAGTACTGCTTGTTCTCTGTGGAG 94 PYGL 202990_at 0.339AGAGAACCTGTTCATCTTTGGCATG 95 OLFML2A 213075_at 0.338CCAATTGTGCTGAGTCTCCTACTAG 96 CYP1B1 202437_s_at 0.336 TAGCCTTTACTGTTTGATATACCAA 97 CAMSAP2 212765_at 0.335GAGCATTGCTTACAGGTTTTTTGTT 98 GOLM1 217771_at 0.335TGGGGCCTAGAAGTTACAGAGCATC 99 IER3 201631_s_at 0.334GAGACTTCGGCGGACCATTAGGAAT 100

TABLE 2 Biomarkers of resistance to irofulven. Dashes indicate that theAffymetrix probeset has not been mapped to a specific gene. AffymetrixIDs refer to the array type HG-U133A. Affymetrix Probe Gene AffymetrixID Correlation Affymetrix Probe Sequence SEQ ID NO: IGJ 212592_at −0.609GCTCACCTGAAAGAGGTATGCTCTC 101 MZB1 221286_s_at −0.608AAAGAAGCTGGGGCTTGCTCTGACG 102 CD8B 207979_s_at −0.59GTAACATTGGGTCCTGGGTCTTTCA 103 ITGB7 205718_at −0.588GAGGATGACGCCAGAGGCACGGTCG 104 CD28 206545_at −0.582GATCAAACTCAGCAGTACTTGGGTG 105 CD53 203416_at −0.573AAAGGGCAAGATCTCATTTCAATTT 106 IQGAP2 203474_at −0.567ACTGTGATATAGGTACTCTGATTTA 107 HCLS1 202957_at −0.554GTACGTCTAGATTGTGTGGTTTGCC 108 CD37 204192_at −0.536CAACGACTCCACAATCCTAGATAAG 109 ICAM3 204949_at −0.527GTACCCCGAGCTGCGGTGTTTGAAG 110 PIM2 204269_at −0.526ACCGGACACCACCAGACAATAGGAT 111 ICAM2 213620_s_at −0.516CTTGGGACATTGCCTTTTCTAGCCC 112 ICAM2 204683_at −0.512CAAACACTCAGCCCCGAAGATGTTG 113 GLYR1 212414_s_at −0.509ATGTGGCTGTTAATATGTGCTTGTT 114 SEPT6 CORO1A 209083_at −0.504GATCGCCAGGTTCTACAAGCTGCAC 115 SLC43A3 213113_s_at −0.498CTTTAGGCCTGTTTGGCTCCGAAGC 116 NFATC3 210555_s_at −0.497TTGTGTGATCCAGCGTCATTTCCAC 117 SSBP2 203787_at −0.493GTATATGTCTGCAATCATGGATAGG 118 PTPN7 204852_s_at −0.492GGGTACAAGCTCCAGAACAGTAACC 119 NCKAP1L 209734_at −0.486GTGGAAGCTGTGGTCACTTTCGCAG 120 RHOH 204951_at −0.474GAGGCTTGGCCACTGGATGTTTTCA 121 MYB 204798_at −0.465TGTGGTTGATAGCCAGTCACTGCCT 122 LCP1 208885_at −0.462TTTGGAGGCAATCCTACCTTGCTTT 123 ARHGAP15 218870_at −0.458ACCTCATGTCCACGCAAAGCTTGGG 124 LRMP 204674_at −0.454AGTGGGATGTCTCTTCAGTTTATGA 125 FARSA 216602_s_at −0.453TGGTGGGCCACAAGGTGAACCTGCA 126 SEPT6 212415_at −0.452TAACCACCGATCAGCACAGCTGTCC 127 LAPTM5 201721_s_at −0.448CATAGGCGAATCTGTTCTGCCCGAG 128 EVI2B 211742_s_at −0.447ACATATACTTATTACCCAGTACAGT 129 USP7 201498_at −0.439GAACATGTACAATTTGCCACTGGGA 130 CCND2 200953_s_at −0.436ATGTTGTATTGGCTATGATGGTGAC 131 SEPT6 213666_at −0.435AAAGGTGTGACCAGTCATTGCCCCT 132 RNASE6 213566_at −0.433ATTTCTAACCCTGCAACTTTTGCCA 133 MYC 202431_s_at −0.432AAAAGCCACAGCATACATCCTGTCC 134 CYTIP 209606_at −0.431AAATCTTAGGTTTGCTTATGCCCAG 135 HBA1 209458_x_at −0.429AGAGAACCCACCATGGTGCTGTCTC 136 HBA2 EVI2A 204774_at −0.428AATTTAATGGTATCTTCCTTGCTGG 137 NARF 219862_s_at −0.428CGGAGAGGTGGTGTTACGCTTTGCT 138 LAPTM5 201720_s_at −0.426AGCTGCTCACAACTGGGTCAACGCT 139 MFNG 204153_s_at −0.426GGTCCCAGCCAATTGTGATGATCCT 140 ARHGDIB 201288_at −0.424GTCACTCAACGTGGTCCCTAGAACA 141 ZFP36L2 201369_s_at −0.422GACGACTGAGGCAAGAGGGCGCCAG 142 MAGEA9 210437_at −0.42GGTTCCTTGTCTATTGGGTGATTTG 143 MAGEA9B NASP 201970_s_at −0.417GTGACTGATATTTCCCACCTTGTCA 144 SMARCC1 201074_at −0.414GAAGAAGGCCTAATCTCTCTCTTTT 145 ARHGAP6 206167_s_at −0.413AAGTGCATAACTATTTTTGACCAGC 146 PTP4A3 206574_s_at −0.413TGGCATCACCGTTGTGGACTGGCCG 147 SAMSN1 220330_s_at −0.411TGGACACATTTTATCCTGATCCACA 148 HBA1 211745_x_at −0.409TGGCGAGTATGGTGCGGAGGCCCTG 149 HBA2 CXCR4 211919_s_at −0.409GTGGTCTATGTTGGCGTCTGGATCC 150 FEN1 204767_s_at −0.408TGGACCTAGACTGTGCTTTTCTGTC 151 HBA1 204018_x_at −0.405CTGACCTCCAAATACCGTTAAGCTG 152 HBA2 HBA1 211699_x_at −0.402CGCACAAGCTTCGGGTGGACCCGGT 153 HBA2 SLC19A1 211576_s_at −0.401GATGTGATGTGAATACTCTTCCCAC 154 ARHGEF3 218501_at −0.399TCATGTGCATTCAAGCTGTGTGACA 155 CXCR4 209201_x_at −0.398AGCACATCATGGTTGGCCTTATCCT 156 UTP3 209486_at −0.397GAAATTCGTCATCTGTTGACACTTA 157 PDSS1 220865_s_at −0.396GCAGCTATCTTACCAGACTGTGCCT 158 MAZ 207824_s_at −0.395AAATGTGAGGCAGCTTTCGCCACGA 159 SELPLG 209879_at −0.395CACTGCACTGCCATTGTCTTTTGGT 160 FARSA 202159_at −0.394TCCTGTGTGGTGTGTCTACTGTGAG 161 LCP2 205269_at −0.394TGTTTTTTACAGCCAAACCTCTGTC 162 STAT5A 203010_at −0.393CCTCCCTCTGAGGCGTGAGGACTCG 163 CISH 221223_x_at −0.392AAAGCCCATCCTGAGACATCTTGCT 164 MAN1A1 221760_at −0.391GGTTTTCATATGAGCTACACATTGT 165 HBA1 217414_x_at −0.388TCAAGGCCGCCTGGGGTAAGGTCGG 166 HBA2 TCF4 222146_s_at −0.388AACAGCTGTATTATCTTAAACCCAC 167 LAIR1 210644_s_at −0.385CAGTTCCCTGGCTGTTTCTAGAGAC 168 MYLIP 220319_s_at −0.382AGCTACGCAAGCTGAAGGAAGCCAT 169 IKZF1 205039_s_at −0.38CGCCCATCCGTGCTATGACATGGAG 170 CXCR4 217028_at −0.379GTATGTCTCGTGGTAGGACTGTAGA 171 TAPBPL 218747_s_at −0.377GAACAGCCTTGGGAGTCATCTTTGC 172 ZFP36L2 201368_at −0.376GTGAAGCAACCGCCTTAGTGCTGAA 173 IL2RG 204116_at −0.374AACCCCAATCCTCTGACAGAAGAAC 174 TNFAIP8 210260_s_at −0.374GTGTCATTGCCTTGAAATGCTTGCT 175 PRKCB 209685_s_at −0.372GAAAAGGGCATTTGGCACCACTCTC 176 PAICS 201014_s_at −0.371GATGTCTGGGAACACTGCATATCCA 177 IKZF1 205038_at −0.371CCCATATCCCTTCTGTAATTTGTAC 178 EZH2 203358_s_at −0.368CTGCCTTAGCTTCAGGAACCTCGAG 179 ADCK3 218168_s_at −0.368CCCTTCCAATCTCTGTTCAGTGCAA 180 SRSF2 214882_s_at −0.365AGTGCAGAGTGCTTGGCTGTTTCCT 181 VARS 201797_s_at −0.364TGAGCTGGCGCTAAGCATCACGCGA 182 NDUFV1 208714_at −0.364GACGGTGCTGATGGACTTCGATGCG 183 REPIN1 219041_s_at −0.363CCTATAATTTCTACCTATTGGGCCT 184 GMFG 204220_at −0.362GGCTGGGGACTGAATTCCTGATGTC 185 MCM7 208795_s_at −0.357GTCAAAGGACTCTCTTCTAGGAGAC 186 TUFM 201113_at −0.356ATGCCCGGGGAGGACCTGAAGTTCA 187 HMHA1 212873_at −0.355CTCCCTTTTCCAGAACACCAGGTGT 188 SON 213538_at −0.355TTGGGTACATAGCCCATTGCCCTTA 189 TXNIP 201008_s_at −0.353TACAAGTTCGGCTTTGAGCTTCCTC 190 NPRL2 203246_s_at −0.353GACTCGGGAAGAGCAGAGCCACCCT 191 TCF4 213891_s_at −0.353GGGAAATTTTTTGCGACTGTACACA 192 NADK 208918_s_at −0.352ATTTTTCCGCAAATCAGTCGGTTGA 193 RRP1B 212846_at −0.352TGTTATCCTTTCTAATTTTTACTGA 194 MAP4K1 214219_x_at −0.352CCAGCAACCTCTACATCCAGGAATG 195 DDX39A 201584_s_at −0.35GCAGTTCAAGGATTTCCAGCGGCGG 196 MAP4K1 206296_x_at −0.35GGTCAGCTTCAGGCCTTCTGGAAGC 197 TXNIP 201009_s_at −0.349GAAGCAGCTTTACCTACTTGTTTCT 198 USP20 203965_at −0.348GCGTCTTTGTTTCTATCAGTCTTTG 199 IFI16 208965_s_at −0.347GACCTGGCTGAAACTCTTAAAAAAG 200

Example 2. Identification of Biomarkers of Sensitivity and Resistance toIrofulven Using Affymetrix HG-U133_Plus_2 Arrays

DNA chip measurements of the 60 cancer cell lines of the NCI60 data setwere also performed using HG-U133_Plus_2 arrays and logit normalized.For each array, the logit transformation was performed followed by aZ-transformation to mean zero and SD 1, and correlated to growthinhibition (log(GI50)). Growth inhibition data of irofulven against thesame cell lines were downloaded from the National Cancer Institute. Eachgene's expression in each cell line was correlated to the growth ofthose cell lines (log(GI50)) in the presence of irofulven. The Pearsoncorrelation coefficient was then determined to identify genes positivelyand negatively correlated to sensitivity to irofulven. Tables 3 and 4show the top positively correlated genes (the biomarkers of sensitivity)and negatively correlated genes (the biomarkers of resistance) using theAffymetrix HG-U133_Plus_2 arrays. The Affymetrix HG-U133_Plus_2 arrayincludes additional genes (e.g., PTGR1 and NME7) that are not includedin the Affymetrix HG-U133A array, which were top ranking biomarkers ofsensitivity and biomarkers of resistance, as shown in Tables 3 and 4.

TABLE 3 Biomarkers of sensitivity to irofulven. Dashes indicate that theAffymetrix probeset has not been mapped to a specific gene. AffymetrixIDs refer to the array type HG-U133_Plus_2. Affymetrix Probe GeneAffymetrix ID Correlation Affymetrix Probe Sequence SEQ ID NO: ATP1B1201242_s_at 0.33 GGACCTACACTTAATCTATATGCTT 201 UCHL1 201387_s_at 0.3AGCCCATGATGCCGTGGCACAGGAA 202 PTGR1 231897_at 0.3AGGATTTGAAAACATGCCAGCTGCA 203 NME7 227556_at 0.29GACACAGCTTTAGAACATACCGGAG 204 PLS3 201215_at 0.27TGTACTCTTGCTTTGTCAAGCTGTT 205 S100A10 200872_at 0.27GGACCTGGACCAGTGTAGAGATGGC 206 CD24 209771_x_at 0.27GAACCTGGTCCTAAGCCTAAAAGTG 207 NQO1 210519_s_at 0.26ATATAGCATTGGGCACACTCCAGCA 208 CD24 216379_x_at 0.26CATGGTCACACACTGATGCTTAGAT 209 PTGR1 228824_s_at 0.26AATGGAGAGGTCCTGCTTGAAGCTT 210 MYOF 201798_s_at 0.26CCAGTAGCAGGTTACGCCATGTCAC 211 LAPTM4B 214039_s_at 0.26ATATTTGATATACTTCTGCCTAACA 212 CALD1 212077_at 0.25ATCACTGCTGACTTTTATTCCAATA 213 PDGFC 218718_at 0.25AAGTAGACATTCAGATCCAGCCATT 214 BASP1 202391_at 0.25TGGATCTCAATGCCAATCCTCCATT 215 NQO1 201468_s_at 0.25AATAGTCAATTTCTACTTCTGGAAG 216 ID1 208937_s_at 0.25CACCCTCAACGGCGAGATCAGCGCC 217 GJA1 201667_at 0.25GAGTGGACTATTAAATGTGCCTAAA 218 ATP1B1 201243_s_at 0.25GTTATGCTTGTATTGAATGCTGTCT 219 CD24 208650_s_at 0.24GAGAGGTTTGACTAGATGATGGATG 220 EGR1 227404_s_at 0.24TGAGCATGTCCCTCACAATTGCACA 221 PERP 222392_x_at 0.24GTGCCACTAAAACAGCCTCAGGAGA 222 CYR61 201289_at 0.24CTTCATTTTGGAGCTTGTGGAGTTG 223 AKR1B10 206561_s_at 0.24GCAACCATACTCAGCTTCAACAGAA 224 PFN2 204992_s_at 0.24TTGCTTCTATTGTTTGGGCCTTGTG 225 TFPI2 209278_s_at 0.23GCTTTCACCTATACTGGCTGTGGAG 226 CTBP2 201218_at 0.23GAGCCTTTGTATAGGTACATTCCTG 227 ASPH 209135_at 0.23AGTAAGTTTTGCTGGATTTTTGTAG 228 PLK2 201939_at 0.23CAGTTCTTGACTTTGGACAATCCCA 229 FAM213A 228155_at 0.22CATATGATGTTTGAGTGCTGTTGTT 230 NQO1 201467_s_at 0.22CATTCTCTGGCCAATTCAGAGTGGC 231 MYOF 211864_s_at 0.22TACCAAATCGACCAGAAACCTCCTT 232 TM4SF1 209386_at 0.22AAAGCCTTTTGTCCTCCAAAGATGA 233 ALCAM 201952_at 0.22GGCAGCCACATGCACGAAGATGCTA 234 WWTR1 202133_at 0.22CCATTTCTTTCACTGATTACACCAT 235 EDIL3 225275_at 0.22ATAACCTTTGCAAACCTTCAAGCTG 236 COL1A1 1556499_s_at 0.22TTTTTATCTTTGACCAACCGAACAT 237 TJP1 202011_at 0.22GATACAATGCTGTGCCCTAAAGTGT 238 PLOD2 202620_s_at 0.22TGTTGACTTGATGTTTTATCACTTC 239 YAP1 224894_at 0.22ACACAGGGAAGTGACTTTGCTACAA 240 EGFR 201983_s_at 0.21GTGTCAACAGCACATTCGACAGCCC 241 MET 203510_at 0.21GCTGAACTGAATGGTACTTCGTATG 242 AJUBA 225806_at 0.21AAAGCTCAATTCCTATTTTGGTGTG 243 CEBPD 203973_s_at 0.21AGAAGCTACAGCCTGGACTTACCAC 244 ASPH 210896_s_at 0.21CGCTCAGTGCTCTTACTAGATGAAC 245 AMIGO2 222108_at 0.21GAGTAACTTCTTAAATCCCTGTTCT 246 AKR1C1 204151_x_at 0.21ATCTCTCCTGCTTGGTGATTTCAGC 247 AKR1C2 209699_x_at 0.21CACATCGCCTCTGGTTAAATCTCTC 248 LOC10193 0400 AKR1C3 209160_at 0.21CAGTCAACTACAGCTGAGTCCATAG 249 LAPTM4B 208767_s_at 0.21TTCACTAACCTTCCCTAGGCATTGA 250 PRSS23 202458_at 0.21AGAATCACTCCTCTCAAATATGCCC 251 CKAP4 200999_s_at 0.21CCATGAAGAGGTTTGCCGTCTGGGC 252 GPX8 227628_at 0.21GAGCACTGTGTATGACTGAAATTTC 253 LOC10192 202237_at 0.21CAACAACGAAGGACTTTTCTCCCTG 254 8916 NNMT LAMC1 200771_at 0.21TCACTTTAGCTGTTTGCCTTACTCT 255 TGFBI 201506_at 0.21AGAAACTTGGATGTCACTGCCTGAC 256 LAPTM4B 208029_s_at 0.2AAGACCATTAGAAAGCACCAGGCCG 257 CD24 266_s_at 0.2CTACTGTGTGTGTGAATGAACACTC 258 PERP 217744_s_at 0.2TTCTACACATCTGCCTAACTTGGGA 259 AKR1C2 211653_x_at 0.2CCCCCCTAATTATCCATTTTCTGAT 260 LOC10193 0400 YAP1 224895_at 0.2GTTTTATGTCCTTGTTCCTAATGTA 261 ABCC3 208161_s_at 0.2CACCCCTAGGAACTCAGTCCTGTAC 262 LIMCH1 212328_at 0.2TGGGTTCTTTGCATGTGGGTTCCAT 263 KRT19 201650_at 0.2GCTGAGCATGAAAGCTGCCTTGGAA 264 CD24 208651_x_at 0.2AACACTTTTTGTTATCAGTCAATAT 265 CRIM1 228496_s_at 0.2TAAAGTTCAGCAACCTCTGTCCAAG 266 SEMA3C 203789_s_at 0.2GTGACTTAATATCTATTCCATTTGT 267 TRIM16 204341_at 0.2ATATGGTGCTGTTCTCTATGTGTTT 268 TSPAN6 209108_at 0.2CTTGGGTCTAAGGCATCCACGACTG 269 PTPN14 226282_at 0.2TCACCATCTCTTATGTTGTTGCCGT 270 NGFRAP1 217963_s_at 0.2CAAGTGGGTCTTGTGTTGCCAGCTT 271 SLPI 203021_at 0.19TCTGTCCTCCTAAGAAATCTGCCCA 272 LGALS3 208949_s_at 0.19CACTTTAACCCACGCTTCAATGAGA 273 CTBP2 210554_s_at 0.19GCACCCCAACGAGCAATAGCAGAGA 274 C19orf33 223631_s_at 0.19TTCTCTTACCGCCATGGAGTTCGAC 275 PLOD2 202619_s_at 0.19CTTAATGTCTGCTCTGAGCCTTAAA 276 PPIC 204517_at 0.19CCCTTCCTCAAGTGGTGCTATTTTA 277 ERRFI1 224657_at 0.19GGTACATTACTGCAATGTTCTCTTA 278 CTBP2 201220_x_at 0.19GTTTGCCTGTGGTAGACACCTGCAC 279 NARR 1555630_a_at 0.19AATGCATTGCATCAACCTACTATAG 280 RAB34 FAM213A 224435_at 0.19AAACCACAGACTTTGGCCTCAGAGA 281 SEPT10 212698_s_at 0.19ATAAAGTACCTTTGAGCATGAGTGT 282 LAPTM4B 1554679_a_at 0.19GGTCCTTATTATTCTTCTGTTTATT 283 PON2 201876_at 0.19GACTACTATGGCTGTTTAGAACTAC 284 CD24 209772_s_at 0.19TGGAACTTCAAGTAACTCCTCCCAG 285 TPM1 210986_s_at 0.19ATTCCTTTCTGATTGGCACACGTGC 286 RAB13 202252_at 0.19ACTAGGGAATTTGCTCCTGTTCTGT 287 AKR1C1 216594_x_at 0.19TGTGGTCCTGGCCAAGAGCTACAAT 288 PTK2 208820_at 0.19AAAGCCATGTTGACTATTTTACAGC 289 LAMB1 201505_at 0.18AAAGCTCAAGAATTAGCAAGACTGG 290 GPRC5A 203108_at 0.18TCCCCAAACTTGCTGTCAATTCCGA 291 PAPSS2 203060_s_at 0.18GATTTCAAGCTGTTCTGAGACATCT 292 LPP 241879_at 0.18TAAACAGCCACAATCGCATTCAGTC 293 GNG11 204115_at 0.18GGGAGAAACTGCATCCTAAGTGGAA 294 DDAH1 209094_at 0.18TATGGTTATCCACTCTGTGTGCCAA 295 CYR61 210764_s_at 0.18GAAATACCGGCCCAAGTACTGCGGT 296 GNG12 212294_at 0.18GTAATAATGTCTTGTTTTTAGCCAT 297 TSPAN6 209109_s_at 0.18ATTTCCTTTGGAGTTGCTTGCTTCC 298 LIMCH1 212327_at 0.18TCCAAAAGCTAGTCCCTACTCTTTA 299 SFN 33323_r_at 0.18CCATGTTTCCTCTCAATAAAGTTCC 300

TABLE 4 Biomarkers of resistance to irofulven. Dashes indicate that theAffymetrix probeset has not been mapped to a specific gene. AffymetrixIDs refer to the array type HG-U133_Plus_2. Affymetrix Probe GeneAffymetrix ID Correlation Affymetrix Probe Sequence SEQ ID NO: IGLC1214677_x_at −0.31 AAGTCCCACAGAAGCTACAGCTGCC 301 IGLC1 209138_x_at −0.29AAGACAGTGGCCCCTACAGAATGTT 302 CYAT1 215121_x_at −0.28GAGCCGTGACAGTGGCCTGGAAGGC 303 IGLC1 IGLV1-44 LAPTM5 201721_s_at −0.26AAAACAGTCCCTTCAAACACACAAT 304 ARHGDIB 201288_at −0.25GTCTCCATCTCAGTACACAATCATT 305 IGLV1-44 215379_x_at −0.25CTCCTCTGAGGAGCTTCAAGCCAAC 306 SLC43A3 213113_s_at −0.24CGAAGCCTATATGTGCCTGGATCCT 307 LCP1 208885_at −0.24TAAGCATCCTTAGGGTTCTGCCTCT 308 HCLS1 202957_at −0.22CTGTCTACTGCAACTGTGATTTCCC 309 CD53 203416_at −0.22AAAGGGCAAGATCTCATTTCAATTT 310 ARHGDIB 1555812_a_at −0.22CTGTGGTGACAGATCCGAAAGCCCC 311 SLC43A3 210692_s_at −0.21TCATCCTGCAAGTGATCAGCCGCTC 312 MZB1 221286_s_at −0.21TAAAACCCAGTGACCTCACTTCTTT 313 RASSF5 223322_at −0.21TGCTGCAACCGCTGTGAATGCTGCT 314 FAM46C 226811_at −0.2TGTTCTATATTTGCATAGCCTTTGA 315 RCSD1 225763_at −0.2CAAAAAGGCCTTATGTCACCACTGG 316 IGJ 212592_at −0.2ATAGGTCAATTATATGTCTCCATTA 317 IGLC1 217148_x_at −0.19TAATCGGCCCTCAGGGGTTTCTAAT 318 IGLJ3 IGLV2-14 IGLV@ LPXN 216250_s_at−0.19 TTTCACCTCTAGTGATGGCCCTACT 319 ITGB7 205718_at −0.19TGCAAAGAGCGGACCCTGGACAACC 320 GTSF1 227711_at −0.19AACTGACAAGCACACTTTTTTCCTC 321 IGLL3P 215946_x_at −0.19GTGGCCCCTGCAGAATGTTCATAGG 322 ICAM2 213620_s_at −0.19TAGTCACGGTGGTGTCGGTGTTGCT 323 PRSS2 205402_x_at −0.18GAGAGCACAACATCGAAGTCCTGGA 324 MZB1 223565_at −0.18TACACGATGTCCTGGACCGGAGCTG 325 IFITM1 214022_s_at −0.18CAACCTTTGCACTCCACTGTGCAAT 326 IKZF1 227346_at −0.18TGCAGTCATATTTCCAGTCTGCCTC 327 CELF2 202157_s_at −0.18GTCACCTTTTGCTGAACTCACAGTT 328 RAC2 213603_s_at −0.18GCCAGATGGTTGCTGCCACAACTTG 329 IFITM1 201601_x_at −0.18ACCCTCTTCTTGAACTGGTGCTGTC 330 IFITM2 PVALB 205336_at −0.18GATGTCGATGACAGACTTGCTGAAC 331 CYFIP2 215785_s_at −0.18CAGCCTGCCATAGGATCCAACTGGA 332 CDK6 224847_at −0.18GTGTTTCTGTTGCAGTGGCAAAGGT 333 MYB 204798_at −0.18AAGCATGCGTTGCACTTCTTTTTTG 334 TCF4 212386_at −0.18GTCTAGTGCTCTTTTGCTATAAAAT 335 CCND2 200953_s_at −0.17TATGTTCATCACCCTTATATCATGT 336 PPM1K 235061_at −0.17AAGACTCTTAGGCAGCTATGGGTTT 337 LAPTM5 201720_s_at −0.17TGTTCAAGTGCGTGTGGCGGTGCTA 338 CDK6 224851_at −0.17GCTTCAGGCAACTTAGCTGTGTACA 339 IFI16 208966_x_at −0.17GACATACTCAATCCTGATTCCAAGT 340 IFI16 206332_s_at −0.17AGAAAGACATACTCAATCCTGATTC 341 EVI2A 204774_at −0.17ACACCAGCTTATCAACCAACACAGC 342 CDK6 224848_at −0.17GGGTGCATGTTCCTTAAAGGTGCAT 343 CORO1A 209083_at −0.17GCTCCAGAAGCGCTTGGACAGGCTG 344 LINC00341 219563_at −0.17GAGGGACCATATCTACTCTTGTCAT 345 SYNE3 LOC10192 205267_at −0.16GCAGGTGTGCTTAGCTCTTGATAGT 346 8620 POU2AF1 CX3CR1 205898_at −0.16GAAAATACCCCATCATTCATGCTAC 347 DOCK2 213160_at −0.16GAAGGCAAACAGATCCCAGACTCGC 348 WIPF1 202664_at −0.16GGTAGAGACTACCCTAGACATCTGC 349 TCF4 213891_s_at −0.16AGGTTGTGTGCTGGAGTTCCTTCAA 350 ST3GAL6 210942_s_at −0.16ACAGGAATTATTGCCATCACATTGG 351 GLYR1 212414_s_at −0.16CAGTGTAGATGGCTCTTGTTTTTGT 352 SEPT6 TRBC1 211796_s_at −0.16CAATGACTCCAGATACTGCCTGAGC 353 AZGP1 209309_at −0.16AAAAAATCATCTCACCTTGAGCCCA 354 LY96 206584_at −0.16ATCTCTATATAACTGTCAACACCAT 355 GIMAP2 232024_at −0.16TGTTTTGTTGCTTACTCTTTAGTAT 356 HERC5 219863_at −0.16ACATCATATTCCTTACCTCTTTTGG 357 IGLL5 217235_x_at −0.15GGCTCCAGGCTGAGGATGAGGCTGA 358 ST3GAL6 213355_at −0.15GGCTCTGGAAATCTAAGTTCATACT 359 ICAM3 204949_at −0.15TGGTACTTATCAGTGCCAAGCGTCC 360 LCP2 205269_at −0.15TGTTTTTTACAGCCAAACCTCTGTC 361 NCKAP1L 238668_at −0.15GAATAGTTGTCTTTTGAGCCTCAGA 362 P2RY8 229686_at −0.15TTGAGTCCGGGTGTTCAAGGGCCAA 363 CCR1 205098_at −0.15CCAAGGACCCCTTATTTATCATGCC 364 GMFG 204220_at −0.15AGCTGAGGAAATTCCGCTTCCGAAA 365 EVI2B 211742_s_at −0.15TCCATCCTCAAATGACTCTTTTTTC 366 - - - 229670_at −0.15GTGAAATCAGGGTGCTGTTACCAAA 367 BCL2 203685_at −0.15GATGGAAAGGCTCGCTCAATCAAGA 368 RAC2 207419_s_at −0.15ATCCACAGACGGACGTCTTCCTCAT 369 IQGAP2 203474_at −0.15TACTGCCTCATGTAAAGACTCTTGC 370 IFI16 208965_s_at −0.15ACTTCATTTTCTTAGCGTTTCTGGA 371 PPM1K 226773_at −0.15CTGATGTGTCCCCAAACTGATTTAA 372 LEF1 221558_s_at −0.15TGTAACACATAGTGGCTTCTCCGCC 373 SLC1A4 209610_s_at −0.15GCTAACCCAGTATGTTCTTCTTTTT 374 CD38 205692_s_at −0.15CTCCTTGACTCCTTGTGGTTTATGT 375 ALDH1A2 207016_s_at −0.15TGAGCCCTGACAGATGCCGTATTTC 376 LOC10192 8635 SSBP2 203787_at −0.15ACATTGACCCACAGGACATTGTAAA 377 PTPRC 212588_at −0.15GCATTTAGTCCAATGTCTTTTTAAG 378 ERN1 227755_at −0.15AGAAAGCATTACCAGTCACCTACTC 379 IRF4 204562_at −0.14TGTGGGAGTCTTGGGTACTCGCACC 380 RHOH 204951_at −0.14CCTCCTGCGTCAATGCCATGGAAGG 381 FAM26F 229390_at −0.14ATACGGTGATTCCTGTTCTTGGCTT 382 IGK IGKC 224795_x_at −0.14CTCGCCCGTCACAAAGAGCTTCAAC 383 PTPRC 207238_s_at −0.14AATGAGGAAACTCCAAACCTCCTGT 384 TCF4 222146_s_at −0.14TATCTTAAACCCACATAAACACTTC 385 RASSF5 1554834_a_at −0.14AGCACATTCCTTTTGCAGGTCTGAG 386 ARHGAP1 218870_at −0.14ACGTTGTCACCGGAGCACTGAAGAT 387 5 LOC10192 8361 - - - 236191_at −0.14GGAACACATTAGTCTATGGGACCTT 388 MAF 209348_s_at −0.14GTATTTGGGACTGAATTGCACTAAG 389 CD8B 215332_s_at −0.14TGTCCCCCTTGTGGAACCAAATGTA 390 LOC10099 6919 ATF3 202672_s_at −0.14GAATTCTCCCAGCGTTAACACAAAA 391 CD3D 213539_at −0.14TGCTCAGTACAGCCACCTTGGAGGA 392 SLC1A4 212811_x_at −0.14GTTTTCATGAACTAGCAACCCCACC 393 IGK IGKC 221651_x_at −0.14CTCGCCCGTCACAAAGAGCTTCAAC 394 IGK IGKC 221671_x_at −0.14AAATCTGGAACTGCCTCTGTTGTGT 395 SAMSN1 220330_s_at −0.14GTATCTCTAACATTCCAAATTACTG 396 PIM2 204269_at −0.14CCCTAGCCTAGGGTCCCATATTGGG 397 DOCK8 225502_at −0.14GTAATGGTGGACTAATTGCTGTATA 398 TCF4 203753_at −0.14GTGCAACTTGAGGGACGACTTTCTT 399 PTPN7 204852_s_at −0.14TCATCCATCTCAGCATCAACACAAT 400

Example 3. Predicting Responsiveness to Irofulven in Various CancerPatient Populations

An mRNA-based predictor of responsiveness to irofulven developedaccording to the methods of the invention was applied to 3,522 patientshaving a variety of cancers. Each patient had a pre-treatmentmeasurement of gene expression with an Affymetrix array. The predictedirofulven sensitivity of each patient was calculated as the differencebetween the mean of the expression levels of the biomarkers ofsensitivity and the mean of the expression levels of the biomarkers ofresistance for the patient. When the patients were grouped by cancertypes, and cancer types predicted to be more responsive to irofulvenwere identified (FIG. 1).

Of 27 different cancer types, solid tumor cancers were predicted to bemore responsive to irofulven treatment than hematological cancers. Inparticular, patients with prostate cancer, ovarian cancer,hepatocellular carcinoma (HCC), breast cancer, cervical cancer, renalcell carcinoma (RCC), esophageal cancer, melanoma, glioma, pancreaticcancer, gastrointestinal stromal tumors (GIST), sarcoma, estrogenreceptor-positive (ERpos) breast cancer, non-small cell lung carcinoma(NSCLC), colon cancer, bladder cancer, and squamous cell carcinoma ofthe head and neck (SCCHN) were predict to be more responsive toirofulven treatment. Patients with hematological cancer types were alsopredicted to be responsive to irofulven treatment, in particular,cutaneous T-cell lymphoma (CTCL), multiple myeloma, acute myelogenousleukemia (AML), acute lympho-blastic leukemia (ALL), chronic lymphocyticleukemia (CLL), myelodysplastic syndrome (MDS), chronic myelogenousleukemia-chronic phase (CMLCP), diffuse large B-cell lymphoma (DLBCL),peripheral T-cell lymphoma (PTCL), and Hodgkin's lymphoma patients werepredicted to be the most responsive to irofulven.

The median of the boxplots shown in FIG. 1 is a cutoff that may be usedto separate patients predicted to be responsive to irofulven treatmentfrom patients predicted to be non-responsive to irofulven treatment fora given cancer type. Values above the median indicate patients predictedto be responsive to irofulven, while values below the median indicatepatients predicted to be non-responsive to irofulven. For a test samplefrom an individual patient, it is useful to compare the test sample tothe reference population for the same cancer type. If the test sample isabove the median for the reference population of the same cancer type,then the patient is predicted to be responsive to irofulven treatment.If the test sample is below the median for the reference population ofthe same cancer type, then the patient is predicted to be non-responsiveto irofulven treatment. This method for predicting patientresponsiveness can also be used when the reference cancer populationconsists of only two patients: a patient responsive to irofulventreatment and a patient non-responsive to irofulven treatment.

Example 4. Predicting Responsiveness of Prostate Cancer Patients toIrofulven

The diagnostic methods of the present invention can be used to predictthe responsiveness of a prostate cancer patient to treatment withirofulven. In particular, the prostate cancer patient may be one thathas not previously received any cancer treatment or one that hasreceived a cancer treatment other than irofulven. Moreover, the patientmay be one diagnosed with prostate cancer or one with recurrence ofprostate cancer. The patient may also have prostate cancer that isresistant to treatment, such as treatment with docetaxel, cabazitaxel,mitoxantrone, estramustine, prednisone, and/or surgery.

A biological sample (e.g., a prostate tissue sample) may be obtainedfrom the patient through methods well known in the art. The sample maybe frozen and/or prepared, e.g., by formalin fixation and paraffinembedding. In particular, mRNA can be isolated from the sample and agene expression profile can be determined, e.g., using a microarrayplatform, such as the Affymetrix HG-U133A or HG-U133_Plus_2 array, forone or more of the biomarkers shown in Tables 1-4. One or more of thebiomarkers shown in Tables 1-4 can also be measured, e.g., by sequencingor PCR-based techniques, such as those described herein.

For example, the expression level of one or more biomarkers ofsensitivity to irofulven can be determined in the sample from thepatient, such as one or more of ATP1B1 (SEQ ID NO: 201 or 219), UCHL1(SEQ ID NO: 202), PTGR1 (SEQ ID NO: 203 or 210), NME7 (SEQ ID NO: 204),PLS3 (SEQ ID NO: 205), S100A10 (SEQ ID NO: 206), CD24 (SEQ ID NO: 207,209, or 220), NQO1 (SEQ ID NO: 208 or 216), MYOF (SEQ ID NO: 211),LAPTM4B (SEQ ID NO: 212), CALD1 (SEQ ID NO: 213), PDGFC (SEQ ID NO:214), BASP1 (SEQ ID NO: 215), ID1 (SEQ ID NO: 217), and GJA1 (SEQ ID NO:218). In particular, the biomarker is ATP1B1 (SEQ ID NO: 201). Theexpression level of one or more biomarkers of resistance to irofulvencan also be determined in the sample from the patient, such as one ormore of IGLC1 (SEQ ID NO: 301, 302, 303, or 318), LAPTM5 (SEQ ID NO: 304or 338), ARHGDIB (SEQ ID NO: 305 or 311), SLC43A3 (SEQ ID NO: 307), LCP1(SEQ ID NO: 308), HCLS1 (SEQ ID NO: 309), CD53 (SEQ ID NO: 310), MZB1(SEQ ID NO: 313), RASSF5 (SEQ ID NO: 314 or 386), FAM46C (SEQ ID NO:315), RCSD1 (SEQ ID NO: 316), IGJ (SEQ ID NO: 317), LPXN (SEQ ID NO:319), ITGB7 (SEQ ID NO: 320), and GTSF1 (SEQ ID NO: 321). In particular,the biomarker is IGLC1 (SEQ ID NO: 301, 302, 303, or 318).

The prostate cancer patient may be responsive to irofulven if theexpression level of one or more of the biomarkers of sensitivity issubstantially similar to the expression level of the biomarkers ofsensitivity in a cell or tissue known to be sensitive to irofulven. Theprostate cancer patient may also be responsive to irofulven if theexpression level of one or more of the biomarkers of resistance issubstantially dissimilar to the expression level of the biomarkers ofresistance in a cell or tissue known to be resistant to irofulven. Ifthe patient is predicted to be responsive, then the patient can beadministered irofulven, such as irofulven administered intravenously ata dose of about 0.05 mg/kg to 5 mg/kg (e.g., about 0.1 mg/kg to 1 mg/kg,such as about 0.45 mg/kg). Conversely, if the patient is predicted to benon-responsive to irofulven treatment, then the patient can beadministered one or more therapies other than irofulven, such asradiation or a therapeutic agent (e.g., docetaxel, cabazitaxel,mitoxantrone, estramustine, prednisone, and/or another therapeutic agentdescribed herein).

Example 5. Predicting Responsiveness of Ovarian Cancer Patients toIrofulven

The diagnostic methods of the present invention can be used to predictthe responsiveness of an ovarian cancer patient to treatment withirofulven. In particular, the ovarian cancer patient may be one that hasnot previously received any cancer treatment or one that has received acancer treatment other than irofulven. Moreover, the patient may be onediagnosed with ovarian cancer or one with recurrence of ovarian cancer.The patient may also have ovarian cancer that is resistant to treatment,such as treatment with docetaxel, carboplatin, bevacizumab, paclitaxel,gemcitabine, doxorubicin, topotecan, etoposide, tamoxifen, and/orletrozole.

A biological sample (e.g., an ovarian tissue sample) may be obtainedfrom the patient through methods well known in the art. The sample maybe frozen and/or prepared, e.g., by formalin fixation and paraffinembedding. In particular, mRNA can be isolated from the sample and agene expression profile can be determined, e.g., using a microarrayplatform, such as the Affymetrix HG-U133A or HG-U133_Plus_2 array, forone or more of the biomarkers shown in Tables 1-4. One or more of thebiomarkers shown in Tables 1-4 can also be measured, e.g., by sequencingor PCR-based techniques, such as those described herein.

For example, the expression level of one or more biomarkers ofsensitivity to irofulven can be determined in the sample from thepatient, such as one or more of ATP1B1 (SEQ ID NO: 201 or 219), UCHL1(SEQ ID NO: 202), PTGR1 (SEQ ID NO: 203 or 210), NME7 (SEQ ID NO: 204),PLS3 (SEQ ID NO: 205), S100A10 (SEQ ID NO: 206), CD24 (SEQ ID NO: 207,209, or 220), NQO1 (SEQ ID NO: 208 or 216), MYOF (SEQ ID NO: 211),LAPTM4B (SEQ ID NO: 212), CALD1 (SEQ ID NO: 213), PDGFC (SEQ ID NO:214), BASP1 (SEQ ID NO: 215), ID1 (SEQ ID NO: 217), and GJA1 (SEQ ID NO:218). In particular, the biomarker is ATP1B1 (SEQ ID NO: 201). Theexpression level of one or more biomarkers of resistance to irofulvencan also be determined in the sample from the patient, such as one ormore of IGLC1 (SEQ ID NO: 301, 302, 303, or 318), LAPTM5 (SEQ ID NO: 304or 338), ARHGDIB (SEQ ID NO: 305 or 311), SLC43A3 (SEQ ID NO: 307), LCP1(SEQ ID NO: 308), HCLS1 (SEQ ID NO: 309), CD53 (SEQ ID NO: 310), MZB1(SEQ ID NO: 313), RASSF5 (SEQ ID NO: 314 or 386), FAM46C (SEQ ID NO:315), RCSD1 (SEQ ID NO: 316), IGJ (SEQ ID NO: 317), LPXN (SEQ ID NO:319), ITGB7 (SEQ ID NO: 320), and GTSF1 (SEQ ID NO: 321). In particular,the biomarker is IGLC1 (SEQ ID NO: 301, 302, 303, or 318).

The ovarian cancer patient may be responsive to irofulven if theexpression level of one or more of the biomarkers of sensitivity issubstantially similar to the expression level of the biomarkers ofsensitivity in a cell or tissue known to be sensitive to irofulven. Theovarian cancer patient may also be responsive to irofulven if theexpression level of one or more of the biomarkers of resistance issubstantially dissimilar to the expression level of the biomarkers ofresistance in a cell or tissue known to be resistant to irofulven. Ifthe patient is predicted to be responsive, then the patient can beadministered irofulven, such as irofulven administered intravenously ata dose of about 0.05 mg/kg to 5 mg/kg (e.g., about 0.1 mg/kg to 1 mg/kg,such as about 0.45 mg/kg). Conversely, if the patient is predicted to benon-responsive to irofulven treatment, then the patient can beadministered one or more therapies other than irofulven, such asradiation or a therapeutic agent (e.g., docetaxel, carboplatin,bevacizumab, paclitaxel, gemcitabine, doxorubicin, topotecan, etoposide,tamoxifen, letrozole, and/or another therapeutic agent describedherein).

Example 6. Predicting Responsiveness of Hepatocellular Carcinoma (HCC)Patients to Irofulven

The diagnostic methods of the present invention can be used to predictthe responsiveness of a hepatocellular carcinoma (HCC) patient totreatment with irofulven. In particular, the HCC patient may be one thathas not previously received any cancer treatment or one that hasreceived a cancer treatment other than irofulven. Moreover, the patientmay be one diagnosed with HCC or one with recurrence of HCC. The patientmay also have HCC that is resistant to treatment, such as treatment withsorafenib, doxorubicin, cisplatin, fluorouracil, gemcitabine,capecitabine, oxaliplatin, interferon-alpha, and/or 5-fluorouracil(5-FU).

A biological sample (e.g., a liver tissue sample) may be obtained fromthe patient through methods well known in the art. The sample may befrozen and/or prepared, e.g., by formalin fixation and paraffinembedding. In particular, mRNA can be isolated from the sample and agene expression profile can be determined, e.g., using a microarrayplatform, such as the Affymetrix HG-U133A or HG-U133_Plus_2 array, forone or more of the biomarkers shown in Tables 1-4. One or more of thebiomarkers shown in Tables 1-4 can also be measured, e.g., by sequencingor PCR-based techniques, such as those described herein.

For example, the expression level of one or more biomarkers ofsensitivity to irofulven can be determined in the sample from thepatient, such as one or more of ATP1B1 (SEQ ID NO: 201 or 219), UCHL1(SEQ ID NO: 202), PTGR1 (SEQ ID NO: 203 or 210), NME7 (SEQ ID NO: 204),PLS3 (SEQ ID NO: 205), S100A10 (SEQ ID NO: 206), CD24 (SEQ ID NO: 207,209, or 220), NQO1 (SEQ ID NO: 208 or 216), MYOF (SEQ ID NO: 211),LAPTM4B (SEQ ID NO: 212), CALD1 (SEQ ID NO: 213), PDGFC (SEQ ID NO:214), BASP1 (SEQ ID NO: 215), ID1 (SEQ ID NO: 217), and GJA1 (SEQ ID NO:218). In particular, the biomarker is ATP1B1 (SEQ ID NO: 201). Theexpression level of one or more biomarkers of resistance to irofulvencan also be determined in the sample from the patient, such as one ormore of IGLC1 (SEQ ID NO: 301, 302, 303, or 318), LAPTM5 (SEQ ID NO: 304or 338), ARHGDIB (SEQ ID NO: 305 or 311), SLC43A3 (SEQ ID NO: 307), LCP1(SEQ ID NO: 308), HCLS1 (SEQ ID NO: 309), CD53 (SEQ ID NO: 310), MZB1(SEQ ID NO: 313), RASSF5 (SEQ ID NO: 314 or 386), FAM46C (SEQ ID NO:315), RCSD1 (SEQ ID NO: 316), IGJ (SEQ ID NO: 317), LPXN (SEQ ID NO:319), ITGB7 (SEQ ID NO: 320), and GTSF1 (SEQ ID NO: 321). In particular,the biomarker is IGLC1 (SEQ ID NO: 301, 302, 303, or 318).

The HCC patient may be responsive to irofulven if the expression levelof one or more of the biomarkers of sensitivity is substantially similarto the expression level of the biomarkers of sensitivity in a cell ortissue known to be sensitive to irofulven. The HCC patient may also beresponsive to irofulven if the expression level of one or more of thebiomarkers of resistance is substantially dissimilar to the expressionlevel of the biomarkers of resistance in a cell or tissue known to beresistant to irofulven. If the patient is predicted to be responsive,then the patient can be administered irofulven, such as irofulvenadministered intravenously at a dose of about 0.05 mg/kg to 5 mg/kg(e.g., about 0.1 mg/kg to 1 mg/kg, such as about 0.45 mg/kg).Conversely, if the patient is predicted to be non-responsive toirofulven treatment, then the patient can be administered one or moretherapies other than irofulven, such as radiation or a therapeutic agent(e.g., docetaxel, carboplatin, bevacizumab, paclitaxel, gemcitabine,doxorubicin, topotecan, etoposide, tamoxifen, letrozole, and/or anothertherapeutic agent described herein).

Other Embodiments

All publications, patents, and patent applications mentioned in theabove specification are hereby incorporated by reference. Variousmodifications and variations of the described device and methods of useof the invention will be apparent to those skilled in the art withoutdeparting from the scope and spirit of the invention. Although theinvention has been described in connection with specific embodiments, itshould be understood that the invention as claimed should not be undulylimited to such specific embodiments. Indeed, various modifications ofthe described modes for carrying out the invention that are obvious tothose skilled in the art are intended to be within the scope of theinvention. For example, it is anticipated that measuring the level ofproteins, metabolites, identifying genetic mutations and DNA copy numbervariations, all will be useful in determining patient responsiveness.

The invention claimed is:
 1. A method of testing a tumor sample of apatient having a known cancer type, wherein the patient is resistant toone or more cancer therapies and has an unknown responsiveness toirofulven, comprising: (a) contacting the sample from the patientcomprising one or more nucleic acid molecules with a device comprising:i) single-stranded nucleic acid molecules capable of specificallyhybridizing with the nucleotides of a plurality of biomarkers ofsensitivity selected from the biomarkers of Tables 1 and 3; and ii)single-stranded nucleic acid molecules capable of specificallyhybridizing with the nucleotides of a plurality of biomarkers ofresistance selected from the biomarkers of Tables 2 and 4; (b) detectinga level of expression of the plurality of biomarkers of sensitivity andthe plurality of biomarkers of resistance by performing microarrayanalysis or quantitative reverse transcriptase polymerase chain reaction(qRT-PCR); and (c) administering irofulven to the patient, wherein thepatient has been determined to be responsive to irofulven.
 2. The methodof claim 1, wherein said method further comprises (c) calculating adifference score for the patient by subtracting the mean expressionlevels of the plurality of biomarkers of resistance from the meanexpression levels of the plurality of biomarkers of sensitivity.
 3. Amethod of treating cancer in a human patient in need thereof comprisingadministering irofulven to the patient, wherein the patient has beendetermined to be responsive to irofulven according to a methodcomprising: (a) contacting a sample from the patient comprising one ormore nucleic acid molecules with a device comprising: i) single-strandednucleic acid molecules capable of specifically hybridizing with thenucleotides of a plurality of biomarkers of sensitivity selected fromthe biomarkers of Tables 1 and 3; and ii) single-stranded nucleic acidmolecules capable of specifically hybridizing with the nucleotides of aplurality of biomarkers of resistance selected from the biomarkers ofTables 2 and 4; and (b) detecting a level of expression of the pluralityof biomarkers of sensitivity and the plurality of biomarkers ofresistance by performing microarray analysis or quantitative reversetranscriptase polymerase chain reaction (qRT-PCR).
 4. The method ofclaim 3, further comprising administering one or more additionaltherapies to the patient prior to, concurrently with, or afteradministration of irofulven.
 5. The method of claim 3, wherein irofulvenis administered by intravenous infusion.
 6. The method of claim 5,comprising administering irofulven to the patient in a treatment regimenat least once per week for at least two weeks and/or on day 1 and day 8of a 3 week treatment regimen.
 7. The method of claim 6, whereinirofulven is administered to the patient at a dose of about 0.2 mg/kg toabout 0.6 mg/kg.
 8. The method of claim 7, wherein irofulven isadministered at a dose of about 0.45 mg/kg.
 9. The method of claim 1,wherein the contacting step (a) and the detecting step (b) occur priorto, concurrent with, or after administration of irofulven to thepatient, and/or wherein the contacting step (a) and the detecting step(b) occur two or more times.
 10. The method of claim 3, wherein thecontacting step (a) and the detecting step (b) occur prior to,concurrent with, or after administration of irofulven to the patient,and/or wherein the contacting step (a) and the detecting step (b) occurtwo or more times during treatment with irofulven.
 11. The method ofclaim 1, wherein one or more of the cancer therapies comprises surgery,radiation, or a therapeutic agent.
 12. The method of claim 1, whereinthe plurality of biomarkers of sensitivity are selected from ATP1B1 (SEQID NO: 201 or 219), UCHL1 (SEQ ID NO: 202), PTGR1 (SEQ ID NO: 203 or210), NME7 (SEQ ID NO: 204), PLS3 (SEQ ID NO: 205), S100A10 (SEQ ID NO:206), CD24 (SEQ ID NO: 207, 209, or 220), NQO1 (SEQ ID NO: 208 or 216),MYOF (SEQ ID NO: 211), LAPTM4B (SEQ ID NO: 212), CALD1 (SEQ ID NO: 213),PDGFC (SEQ ID NO: 214), BASP1 (SEQ ID NO: 215), ID1 (SEQ ID NO: 217),and GJA1 (SEQ ID NO: 218) and/or the plurality of biomarkers ofresistance are selected from IGLC1 (SEQ ID NO: 301, 302, 303, or 318),LAPTM5 (SEQ ID NO: 304 or 338), ARHGDIB (SEQ ID NO: 305 or 311), SLC43A3(SEQ ID NO: 307), LCP1 (SEQ ID NO: 308), HCLS1 (SEQ ID NO: 309), CD53(SEQ ID NO: 310), MZB1 (SEQ ID NO: 313), RASSF5 (SEQ ID NO: 314 or 386),FAM46C (SEQ ID NO: 315), RCSD1 (SEQ ID NO: 316), IGJ (SEQ ID NO: 317),LPXN (SEQ ID NO: 319), ITGB7 (SEQ ID NO: 320), and GTSF1 (SEQ ID NO:321).
 13. The method of claim 1, wherein the device is a microarray. 14.The method of claim 1, wherein the device is for performing a qRT-PCRreaction.
 15. The method of claim 1, wherein the level of expression ofthe plurality of biomarkers of sensitivity is determined by detectingthe level of mRNA transcribed from genes encoding the plurality ofbiomarkers of sensitivity.
 16. The method of claim 1, wherein the levelof expression of the plurality of biomarkers of resistance is determinedby detecting the level of mRNA transcribed from genes encoding theplurality of biomarkers of resistance.
 17. The method of claim 1,wherein the cancer is selected from a solid tumor cancer and ahematological cancer.
 18. The method of claim 1, wherein the cancer isselected from the group consisting of prostate cancer, ovarian cancer,hepatocellular carcinoma (HCC), cervical cancer, renal cell carcinoma(RCC), esophageal cancer, melanoma, glioma, pancreatic cancer,gastrointestinal stromal tumors (GIST), sarcoma, estrogenreceptor-positive (ERpos) breast cancer, non-small cell lung carcinoma(NSCLC), colon cancer, bladder cancer, squamous cell carcinoma of thehead and neck (SCCHN), acute myelogenous leukemia (AML), acutelympho-blastic leukemia (ALL), chronic lymphocytic leukemia (CLL),myelodysplastic syndrome (MDS), chronic myelogenous leukemia-chronicphase (CMLCP), diffuse large B-cell lymphoma (DLBCL), cutaneous T-celllymphoma (CTCL), peripheral T-cell lymphoma (PTCL), and Hodgkin'slymphoma.
 19. The method of claim 3, wherein: (a) the sample from thepatient is a tumor sample; (b) the patient exhibits cancer relapse aftertreatment with a therapeutic agent other than irofulven; (c) the patientexhibits cancer relapse prior to treatment with irofulven; or (d) thepatient has not previously been administered a treatment for cancer. 20.The method of claim 1, wherein irofulven is administered at a dose ofabout 0.2 mg/kg to about 0.6 mg/kg.
 21. The method of claim 4, whereinone or more of the additional therapies comprises surgery, radiation, ora therapeutic agent.
 22. The method of claim 21, wherein the therapeuticagent is selected from the group consisting of docetaxel, cabazitaxel,mitoxantrone, estramustine, prednisone, carboplatin, bevacizumab,paclitaxel, gemcitabine, doxorubicin, topotecan, etoposide, tamoxifen,letrozole, sorafenib, fluorouracil, capecitabine, oxaliplatin,interferon-alpha, 5-fluorouracil (5-FU), a histone deacetylase (HDAC)inhibitor, ipilimumab, bortezomib, carfilzomib, thalidomide,lenalidomide, pomalidomide, dexamethasone, cyclophosphamide,vincristine, melphalan, tegafur, irinotecan, cetuximab, leucovorin,SN-38, everolimus, temsirolimus, bleomycin, lomustine, depsipeptide,erlotinib, cisplatin, busulfan, epirubicin, arsenic trioxide,bendamustine, fulvestrant, teniposide, adriamycin, decitabine,estramustine, azaguanine, aclarubicin, mitomycin, paclitaxel, taxotere,APO010, ara-c, methylprednisolone, methotrexate, methyl-gag, belinostat,idarubicin, IL4-PR38, valproic acid, all-trans retinoic acid (ATRA),cytoxan, suberoylanilide hydroxamic acid, leukeran, fludarabine,vinblastine, dacarbazine, hydroxyurea, tegafur, daunorubicin,mechlorethamine, streptozocin, carmustine, mercaptopurine, dactinomycin,tretinoin, ifosfamide, floxuridine, thioguanine, PSC 833, herceptin,celecoxib, iressa, anastrozole, and rituximab.
 23. The method of claim6, wherein the treatment regimen is repeated two to twenty times. 24.The method of claim 3, wherein the plurality of biomarkers ofsensitivity are selected from two or more of ATP1B1 (SEQ ID NO: 201 or219), UCHL1 (SEQ ID NO: 202), PTGR1 (SEQ ID NO: 203 or 210), NME7 (SEQID NO: 204), PLS3 (SEQ ID NO: 205), S100A10 (SEQ ID NO: 206), CD24 (SEQID NO: 207, 209, or 220), NQO1 (SEQ ID NO: 208 or 216), MYOF (SEQ ID NO:211), LAPTM4B (SEQ ID NO: 212), CALD1 (SEQ ID NO: 213), PDGFC (SEQ IDNO: 214), BASP1 (SEQ ID NO: 215), ID1 (SEQ ID NO: 217), and GJA1 (SEQ IDNO: 218) and/or the plurality of biomarkers of resistance are selectedfrom two or more of IGLC1 (SEQ ID NO: 301, 302, 303, or 318), LAPTM5(SEQ ID NO: 304 or 338), ARHGDIB (SEQ ID NO: 305 or 311), SLC43A3 (SEQID NO: 307), LCP1 (SEQ ID NO: 308), HCLS1 (SEQ ID NO: 309), CD53 (SEQ IDNO: 310), MZB1 (SEQ ID NO: 313), RASSF5 (SEQ ID NO: 314 or 386), FAM46C(SEQ ID NO: 315), RCSD1 (SEQ ID NO: 316), IGJ (SEQ ID NO: 317), LPXN(SEQ ID NO: 319), ITGB7 (SEQ ID NO: 320), and GTSF1 (SEQ ID NO: 321).25. The method of claim 3, wherein the cancer is selected from a solidtumor cancer and a hematological cancer.
 26. The method of claim 3,wherein the cancer is selected from the group consisting of prostatecancer, ovarian cancer, hepatocellular carcinoma (HCC), cervical cancer,renal cell carcinoma (RCC), esophageal cancer, melanoma, glioma,pancreatic cancer, gastrointestinal stromal tumors (GIST), sarcoma,estrogen receptor-positive (ERpos) breast cancer, non-small cell lungcarcinoma (NSCLC), colon cancer, bladder cancer, squamous cell carcinomaof the head and neck (SCCHN), acute myelogenous leukemia (AML), acutelympho-blastic leukemia (ALL), chronic lymphocytic leukemia (CLL),myelodysplastic syndrome (MDS), chronic myelogenous leukemia-chronicphase (CMLCP), diffuse large B-cell lymphoma (DLBCL), cutaneous T-celllymphoma (CTCL), peripheral T-cell lymphoma (PTCL), and Hodgkin'slymphoma.
 27. The method of claim 2, wherein: i) the difference score issubstantially similar to a difference score of a patient known to besensitive to irofulven; and/or ii) the difference score is substantiallydissimilar to a difference score of a patient known to be resistant toirofulven.
 28. The method of claim 3, wherein said method furthercomprises (c) calculating a difference score for the patient bysubtracting the mean expression levels of the plurality of biomarkers ofresistance from the mean expression levels of the plurality ofbiomarkers of sensitivity.
 29. The method of claim 28, wherein: i) thedifference score is substantially similar to a difference score of apatient known to be sensitive to irofulven; and/or ii) the differencescore is substantially dissimilar to a difference score of a patientknown to be resistant to irofulven.